Photoreactive synthetic regulator of protein function and methods of use thereof

ABSTRACT

The present disclosure provides a photoreactive synthetic regulator of protein function. The present disclosure further provides a light-regulated polypeptide that includes a subject synthetic regulator. Also provided are cells and membranes comprising a subject light-regulated polypeptide. The present disclosure further provides methods of modulating protein function, involving use of light.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.13/059,052, filed Feb. 11, 2011, now U.S. Pat. No. 8,993,736, which is anational stage filing under 35 U.S.C. §371 of PCT/US2009/062491, filedOct. 29, 2009, which claims the benefit of U.S. Provisional PatentApplication No. 61/110,369, filed Oct. 31, 2008, and U.S. ProvisionalPatent Application No. 61/122,608, filed Dec. 15, 2008, whichapplications are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No.1PN2-EY018241 awarded by the National Institutes of Health, and GrantNo. CHE 0724212 awarded by the National Science Foundation. Thegovernment has certain rights in the invention.

BACKGROUND

The precise regulation of protein activity is fundamental to life. Amechanism of regulation, found across protein classes, from enzymes, tomotors, to signaling proteins, is allosteric control of an active siteby a remote regulatory binding site.

Many proteins function like molecular machines that undergo mechanicalmovements in response to input signals. These signals can consist ofchanges in voltage, membrane tension, temperature or, most commonly,ligand concentration. Ligands provide information about events in theexternal world, or about the energetic or biosynthetic state of thecell, and can be as small as a proton or as large as a whole protein. Inallostery, ligand binding induces a structural change of a sensordomain, which propagates to a functional domain of the protein andalters its behavior. Such conformational control can operate over longdistances, crossing a membrane or passing from one protein to another ina complex.

Photochromic molecules have emerged as powerful optical tools to controlprotein and cellular function in neuroscience. Photoswitchable tetheredligands are covalently anchored to protein surfaces throughphotoisomerizable tethers. Photoswitching changes the length andgeometry of the tether to alter the effective concentration of ligand atits binding site, thereby modulating protein function.

There is a need in the art for methods of regulating protein function.

Literature

Gorostiza and Isacoff (2008) Science 322:395; Kaufman et al. (1968)Science 162:1487-1489; Bartels et al. (1971) Proc. Natl. Acad. Sci.U.S.A. 68:1820-1823; Fujita et al. (2006) Biochemistry 45:6581-6586;Caamano et al. (2000) Angew. Chem., Int. Ed. Engl. 39:3104-3107; Mayerand Heckel (2006) Angew. Chem., Int. Ed. Engl. 45:4900-4921; Givens etal. (1998) In Methods in Enzymology, Marriott, G., Ed. Academic Press,New York, 291:1-29; Volgraf et al. (2006) Nature Chem. Biol. 2:47-52;U.S. Patent Publication No. 2007/0128662; Lester et al. J. Gen. Physiol.75:207-232 (1980); Banghart et al. Nature Neurosci. 7:1381-1386 (2004);WO 2007/024290.

SUMMARY OF THE INVENTION

The present disclosure provides a photoreactive synthetic regulator ofprotein function. The present disclosure further provides alight-regulated polypeptide that includes a subject synthetic regulator.Also provided are cells and membranes comprising a subjectlight-regulated polypeptide. The present disclosure further providesmethods of modulating protein function, involving use of light.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C depict of modes of photoregulating potassium channels.

FIGS. 2A-C depict: (a) chemical structures of AAQ and EtAcAQ and (b, c)voltage-gated steady-state currents from Shaker IR channels afterblocker treatment.

FIGS. 3A-F depict the effect of AAQ on the Sh-IR internal TEA-bindingsite.

FIG. 4 provides a table of structure, potency and photoeffect ofphotochromic Azo-QAs on Shaker IR.

FIGS. 5A and 5B depict BzAQ photoregulation of endogenous K⁺ channels indissociated hippocampal neurons to modulate neural activity.

FIG. 6 depicts the activity of QAQ as a photochromic blocker forvoltage-gated cation channels.

FIG. 7 depicts the activity of QAQ as a silencer of neuronal activity.

FIG. 8 schematically depicts use of an agonist of a ligand-gatednon-selective cation channel to load membrane-impermeant QAQ into cells.

FIG. 9 depicts loading of QAQ into cells through non-selective ionchannels.

FIG. 10 depicts loading of QAQ into hippocampal neurons transfected withthe non-selective cation channel P2X₇R, using ATP (left panel); andloading of QAQ into sensory neurons that naturally express thenon-selective cation channel TRPV1.

FIG. 11 depicts in vivo blocking of capsaicin-activated sensory neurons.

DEFINITIONS

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

The following terms have the following meanings unless otherwiseindicated. Any undefined terms have their art recognized meanings.

The term “alkyl” refers to a monoradical branched or unbranchedsaturated hydrocarbon chain, e.g., having from 1 to 40 carbon atoms,from 1 to 10 carbon atoms, or from 1 to 6 carbon atoms. This term isexemplified by groups such as methyl, ethyl, n-propyl, iso-propyl,n-butyl, iso-butyl, n-hexyl, n-decyl, tetradecyl, and the like.

The term “substituted alkyl” refers to an alkyl group as defined abovewherein one or more carbon atoms in the alkyl chain have been optionallyreplaced with a heteroatom such as —O—, —S(O)_(n)—(where n is 0 to 2),—NR— (where R is hydrogen or alkyl) and having from 1 to 5 substituentsselected from the group consisting of alkoxy, substituted alkoxy,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-aryl,—SO₂-heteroaryl, and —NR^(a)Rb, wherein R^(a) and R^(b) may be the sameor different and are chosen from hydrogen, optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl andheterocyclic.

The term “alkylaminoalkyl”, “alkylaminoalkenyl” and “alkylaminoalkynyl”refers to the groups R^(a)NIIR^(b)— where R^(a) is alkyl group asdefined above and R^(b) is alkylene, alkenylene or alkynylene group asdefined above.

The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl and-substituted alkylene-aryl where alkylene, substituted alkylene and arylare defined herein.

The term “alkoxy” refers to the groups alkyl-O—, alkenyl-O—,cycloalkyl-O—, cycloalkenyl-O—, and alkynyl-O—, where alkyl, alkenyl,cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

The term “substituted alkoxy” refers to the groups substituted alkyl-O—,substituted alkenyl-O—, substituted cycloalkyl-O—, substitutedcycloalkenyl-O—, and substituted alkynyl-O— where substituted alkyl,substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyland substituted alkynyl are as defined herein.

The term “haloalkoxy” refers to the groups alkyl-O— wherein one or morehydrogen atoms on the alkyl group have been substituted with a halogroup and include, by way of examples, groups such as trifluoromethoxy,and the like.

The term “alkylalkoxy” refers to the groups -alkylene-O-alkyl,alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, andsubstituted alkylene-O-substituted alkyl wherein alkyl, substitutedalkyl, alkylene and substituted alkylene are as defined herein.

The term “alkylthioalkoxy” refers to the group -alkylene-S-alkyl,alkylene-S-substituted alkyl, substituted alkylene-S-alkyl andsubstituted alkylene-S-substituted alkyl wherein alkyl, substitutedalkyl, alkylene and substituted alkylene are as defined herein.

The term “alkenyl” refers to a monoradical of a branched or unbranchedunsaturated hydrocarbon group having from 2 to 40 carbon atoms, from 2to 10 carbon atoms, or from 2 to 6 carbon atoms and having at least 1site (e.g., from 1-6 sites) of vinyl unsaturation.

The term “substituted alkenyl” refers to an alkenyl group as definedabove having from 1 to 5 substituents, or from 1 to 3 substituents,selected from alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

The term “alkynyl” refers to a monoradical of an unsaturated hydrocarbonhaving from 2 to 40 carbon atoms, from 2 to 20 carbon atoms, or from 2to 6 carbon atoms and having at least 1 site (e.g., from 1-6 sites) ofacetylene (triple bond) unsaturation.

The term “substituted alkynyl” refers to an alkynyl group as definedabove having from 1 to 5 substituents, or from 1 to 3 substituents,selected from alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,alkyl, —SO₂-substituted alkyl, —SO₂-aryl, and —SO₂-heteroaryl.

The term “acyl” refers to the groups HC(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, cycloalkyl-C(O)—, substituted cycloalkyl-C(O)—,cycloalkenyl-C(O)—, substituted cycloalkenyl-C(O)—, aryl-C(O)—,heteroaryl-C(O)— and heterocyclic-C(O)— where alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, heteroaryl, and heterocyclic are as defined herein.

The term “acylamino” or “aminocarbonyl” refers to the group —C(O)NRRwhere each R is independently hydrogen, alkyl, substituted alkyl, aryl,heteroaryl, heterocyclic or where both R groups are joined to form aheterocyclic group (e.g., morpholino) wherein alkyl, substituted alkyl,aryl, heteroaryl, and heterocyclic are as defined herein.

The term “aminoacyl” refers to the group —NRC(O)R where each R isindependently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, orheterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl, andheterocyclic are as defined herein.

The term “aminoacyloxy” or “alkoxycarbonylamino” refers to the group—NRC(O)OR where each R is independently hydrogen, alkyl, substitutedalkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substitutedalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.

The term “acyloxy” refers to the groups alkyl-C(O)O—, substitutedalkyl-C(O)O—, cycloalkyl-C(O)O—, substituted cycloalkyl-C(O)O—,aryl-C(O)O—, heteroaryl-C(O)O—, and heterocyclic-C(O)O— wherein alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl,and heterocyclic are as defined herein.

The term “aryl” refers to an unsaturated aromatic carbocyclic group offrom 6 to 20 carbon atoms having a single ring (e.g., phenyl) ormultiple condensed (fused) rings (e.g., naphthyl or anthryl). Exemplaryaryls include phenyl, naphthyl and the like. Unless otherwiseconstrained by the definition for the aryl substituent, such aryl groupscan optionally be substituted with from 1 to 5 substituents, or from 1to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl,alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl,substituted alkoxy, substituted alkenyl, substituted alkynyl,substituted cycloalkyl, substituted cycloalkenyl, amino, substitutedamino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl,carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy,heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy,substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl and trihalomethyl.

The term “aryloxy” refers to the group aryl-O— wherein the aryl group isas defined above including optionally substituted aryl groups as alsodefined herein.

The term “amino” refers to the group —NH₂.

The term “substituted amino” refers to the group —NRR where each R isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,substituted alkynyl, aryl, heteroaryl, and heterocyclic provided thatboth R's are not hydrogen.

The term “carboxyalkyl” or “alkoxycarbonyl” refers to the groups“—C(O)O-alkyl”, “—C(O)O— substituted alkyl”, “—C(O)O-cycloalkyl”,“—C(O)O-substituted cycloalkyl”, “—C(O)O-alkenyl”, “—C(O)O— substitutedalkenyl”, “—C(O)O-alkynyl” and “—C(O)O-substituted alkynyl” where alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, alkynyl and substituted alkynyl alkynyl are asdefined herein.

The term “cycloalkyl” refers to cyclic alkyl groups of from 3 to 20carbon atoms having a single cyclic ring or multiple condensed rings.Such cycloalkyl groups include, by way of example, single ringstructures such as cyclopropyl, cyclobutyl, cyclopentyl, cycloodyl, andthe like, or multiple ring structures such as adamantanyl, and the like.

The term “substituted cycloalkyl” refers to cycloalkyl groups havingfrom 1 to 5 substituents, or from 1 to 3 substituents, selected fromalkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano,halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy,thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

The term “cycloalkenyl” refers to cyclic alkenyl groups of from 4 to 20carbon atoms having a single cyclic ring and at least one point ofinternal unsaturation. Examples of suitable cycloalkenyl groups include,for instance, cyclobut-2-enyl, cyclopent-3-enyl, cyclooct-3-enyl, andthe like.

The term “substituted cycloalkenyl” refers to cycloalkenyl groups havingfrom 1 to 5 substituents, or from 1 to 3 substituents, selected fromalkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano,halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy,thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

The term “halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

The term “heteroaryl” refers to an aromatic group of from 1 to 15 carbonatoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfurwithin at least one ring (if there is more than one ring). Unlessotherwise constrained by the definition for the heteroaryl substituent,such heteroaryl groups can be optionally substituted with 1 to 5substituents, or from 1 to 3 substituents, selected from acyloxy,hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, substituted alkyl, substituted alkoxy, substitutedalkenyl, substituted alkynyl, substituted cycloalkyl, substitutedcycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl,aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro,heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy,oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy,thioheteroaryloxy, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl,and trihalomethyl.

The term “heteroaralkyl” refers to the groups -alkylene-heteroaryl wherealkylene and heteroaryl are defined herein. Such heteroaralkyl groupsare exemplified by pyridylmethyl, pyridylethyl, indolylmethyl, and thelike.

The term “heteroaryloxy” refers to the group heteroaryl-O—.

The term “heterocycle” or “heterocyclic” refers to a monoradicalsaturated or unsaturated group having a single ring or multiplecondensed rings, from 1 to 40 carbon atoms and from 1 to 10 heteroatoms, e.g., from 1 to 4 heteroatoms, selected from nitrogen, sulfur,phosphorus, and/or oxygen within the ring. Unless otherwise constrainedby the definition for the heterocyclic substituent, such heterocyclicgroups can be optionally substituted with 1 to 5, or from 1 to 3substituents, selected from alkoxy, substituted alkoxy, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

Examples of nitrogen heteroaryls and heterocycles include, but are notlimited to, pyrrole, thiophene, furan, imidazole, pyrazole, pyridine,pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, pyrrolidine, piperidine, piperazine,indoline, morpholine, tetrahydrofuranyl, tetrahydrothiophene, and thelike as well as N-alkoxy-nitrogen containing heterocycles.

The term “heterocyclooxy” refers to the group heterocyclic-O—.

The term “heterocyclothio” refers to the group heterocyclic-S—.

The term “heterocyclene” refers to the diradical group formed from aheterocycle, as defined herein, and is exemplified by the groups2,6-morpholino, 2,5-morpholino and the like.

The term “heteroarylamino” refers to a 5 membered aromatic ring whereinone or two ring atoms are N, the remaining ring atoms being C. Theheteroarylamino ring may be fused to a cycloalkyl, aryl or heteroarylring, and it may be optionally substituted with one or moresubstituents, e.g., one or two substituents, selected from alkyl,substituted alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl,halo, cyano, acyl, amino, substituted amino, acylamino, —OR (where R ishydrogen, alkyl, alkenyl, cycloalkyl, acyl, aryl, heteroaryl, aralkyl,or heteroaralkyl), or —S(O)_(n)R where n is an integer from 0 to 2 and Ris hydrogen (provided that n is 0), alkyl, alkenyl, cycloalkyl, amino,heterocyclo, aryl, heteroaryl, aralkyl, or heteroaralkyl.

The term “heterocycloamino” refers to a saturated monovalent cyclicgroup of 4 to 8 ring atoms, wherein at least one ring atom is N andoptionally contains one or two additional ring heteroatoms selected fromthe group consisting of N, O, or S(O)n (where n is an integer from 0 to2), the remaining ring atoms being C, where one or two C atoms mayoptionally be replaced by a carbonyl group. The heterocycloamino ringmay be fused to a cycloalkyl, aryl or heteroaryl ring, and it may beoptionally substituted with one or more substituents, e.g., one or twosubstituents, selected from alkyl, substituted alkyl, cycloalkyl, aryl,aralkyl, heteroaryl, heteroaralkyl, halo, cyano, acyl, amino,substituted amino, acylamino, —OR (where R is hydrogen, alkyl, alkenyl,cycloalkyl, acyl, aryl, heteroaryl, aralkyl, or heteroaralkyl), or—S(O)_(n)R [where n is an integer from 0 to 2 and R is hydrogen(provided that n is 0), alkyl, alkenyl, cycloalkyl, amino, heterocyclo,aryl, heteroaryl, aralkyl, or heteroaralkyl].

The term “oxyacylamino” or “aminocarbonyloxy” refers to the group—OC(O)NRR where each R is independently hydrogen, alkyl, substitutedalkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substitutedalkyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term “thiol” refers to the group —SH.

The term “thioalkoxy” or “alkylthio” refers to the group —S-alkyl.

The term “substituted thioalkoxy” refers to the group —S-substitutedalkyl.

The term “thioaryloxy” refers to the group aryl-S— wherein the arylgroup is as defined above including optionally substituted aryl groupsalso defined above.

The term “thioheteroaryloxy” refers to the group heteroaryl-S— whereinthe heteroaryl group is as defined above including optionallysubstituted aryl groups as also defined above.

As to any of the above groups which contain one or more substituents, itis understood, of course, that such groups do not contain anysubstitution or substitution patterns which are sterically impracticaland/or synthetically non-feasible. In addition, the compounds of theembodiments include all stereochemical isomers arising from thesubstitution of these compounds.

The term “pharmaceutically-acceptable salt” refers to salts which retainbiological effectiveness and are not biologically or otherwiseundesirable. In many cases, the compounds of the embodiments are capableof forming acid and/or base salts by virtue of the presence of aminoand/or carboxyl groups or groups similar thereto.

Pharmaceutically-acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines, such asalkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines,di(substituted alkyl)amines, tri(substituted alkyl)amines, alkenylamines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines,di(substituted alkenyl)amines, tri(substituted alkenyl)amines,cycloalkyl amines, di(cycloalkyl)amines, tri(cycloalkyl)amines,substituted cycloalkyl amines, disubstituted cycloalkyl amine,trisubstituted cycloalkyl amines, cycloalkenyl amines,di(cycloalkenyl)amines, tri(cycloalkenyl)amines, substitutedcycloalkenyl amines, disubstituted cycloalkenyl amine, trisubstitutedcycloalkenyl amines, aryl amines, diaryl amines, triaryl amines,heteroaryl amines, diheleroaryl amines, triheteroaryl amines,heterocyclic amines, diheterocyclic amines, triheterocyclic amines,mixed di- and tri-amines where at least two of the substituents on theamine are different and are selected from the group consisting of alkyl,substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl,heterocyclic, and the like. Also included are amines where the two orthree substituents, together with the amino nitrogen, form aheterocyclic or heteroaryl group. Examples of suitable amines include,by way of example only, isopropylamine, trimethyl amine, diethyl amine,tri(iso-propyl)amine, tri(n-propyl)amine, ethanolamine,2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine,caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,glucosamine, N-alkylglucamines, theobromine, purines, piperazine,piperidine, morpholine, N-ethylpiperidine, and the like.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “asynthetic regulator of protein function” includes a plurality of suchregulators and reference to “the ligand” includes reference to one ormore ligands and equivalents thereof known to those skilled in the art,and so forth. It is further noted that the claims may be drafted toexclude any optional element. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely,” “only” and the like in connection with the recitation of claimelements, or use of a “negative” limitation.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides a photoreactive synthetic regulator ofprotein function. The present disclosure further provides alight-regulated polypeptide that includes a subject synthetic regulator.Also provided are cells and membranes comprising a subjectlight-regulated polypeptide. The present disclosure further providesmethods of modulating protein function, involving use of light.

Synthetic Regulator of Protein Function

The present disclosure provides a synthetic regulator of proteinfunction. A subject synthetic regulator of protein function is usefulfor regulating protein function by use of light. A subject syntheticprotein regulator comprises: a) a polypeptide association moiety, whichcan provide for interaction with the polypeptide or occlusion to a pore;b) a photoisomerizable group; and c) a ligand that binds to a ligandbinding site (e.g., an active site, an allosteric site, a pore of an ionchannel, etc.) of a protein. A subject synthetic protein regulator (alsoreferred to herein as a “synthetic regulator,” or “a photoswitch”) issuitable for attachment to a variety of polypeptides, includingnaturally-occurring (native, or endogenous) polypeptides, recombinantpolypeptides, synthetic polypeptides, etc.

A subject synthetic regulator can be provided in any number ofconfigurations, including linear and branched. In some embodiments, asubject synthetic regulator has the structure:(A)_(n)-X₁—(B)_(m)—X₂—(C)_(p), where A is a polypeptide associationmoiety, B is a photoisomerizable group, and C is a ligand, and whereeach of n, m, and p is independently 1 to 10, e.g., where each of n, m,and p is independently one, two, three, four, five, six, seven, eight,nine, or ten, and where X₁, when present, is a spacer; and X₂, whenpresent, is a spacer. In some embodiments, X₁ and X₂ are not present. Insome embodiments, X₁ and X₂ are both present. In some embodiments, onlyone of X₁ and X₂ is present. In some embodiments, X₁ and X₂ areindependently selected from alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, acyl, acylamino, andaminoacyl. In some embodiments, each of n, m, and p is 1. In otherembodiments, a subject synthetic regulator comprises two or more (e.g.,2 to 10, e.g., two, three, four, five, six, seven, eight, nine, or ten)photoisomerizable groups. In some embodiments, where the syntheticregulator comprises two or more photoisomerizable groups, the two ormore photoisomerizable groups are arranged in tandem, either directly orseparated by a spacer.

A subject synthetic regulator can be provided in any number ofconfigurations, including linear and branched. In some embodiments, asubject synthetic regulator has the structure: (A)_(n)-(B)_(m)—(C)_(p),where A is a polypeptide association moiety, B is a photoisomerizablegroup, and C is a ligand, and where each of n, m, and p is independently1 to 10, e.g., where each of n, m, and p is independently one, two,three, four, five, six, seven, eight, nine, or ten. In some embodiments,each of n, m, and p is 1, e.g., a subject synthetic regulator has thestructure A-B-C.

In other embodiments, a subject synthetic regulator has the structure:C—X₁(A)-B—X₂(A)-C, where A is a polypeptide association moiety, B is aphotoisomerizable group, and C is a ligand, where X₁, when present, is aspacer, where X₂, when present, is a spacer, and where X(A) indicatesthat A branches off of X. Suitable spacers include peptide spacers(e.g., spacers of from about 1 to about 20 amino acids in length);non-peptide spacers, e.g., non-peptide polymers of various numbers ofmonomeric units, e.g., from one to about 20 units. In these embodiments,B can be present in multiple copies, either directly or in tandem.

Protein Association Moiety

The protein association moiety can be any of a variety of functionalgroups that provide for association of the synthetic regulator with apolypeptide or occlusion to an opening of a pore. In some embodiments,the protein association moiety can provide for association with an aminoacid side chain in a polypeptide. In some embodiments, the proteinassociation moiety can provide for association with a ligand-bindingpolypeptide, and with a membrane component. In other embodiments, theprotein association moiety can provide for association of the syntheticregulator with a sugar residue in the polypeptide. In other embodiments,the protein association moiety can provide for association of thesynthetic regulator with a moiety other than a sugar residue or an aminoacid side chain. In some embodiments, the protein association moiety cancomprise a reactive electrophile that can provide for association withan amino acid in the ligand-binding polypeptide. In some embodiments,the protein association moiety can comprise a reactive electrophile thatcan provide for association with an amino acid at or near aligand-binding site in a ligand-binding protein. In some embodiments,the protein association moiety can provide for occlusion to an openingof a pore, such as an ion channel.

Association of the synthetic regulator with a polypeptide includesnon-covalent associations such as ionic interactions, van der Waalsinteractions, hydrogen bonding, and the like. The association is ahigh-affinity association, e.g., the association between the syntheticregulator and the polypeptide has an affinity of from about 10⁻³M toabout 10⁻¹² M, or greater than 10⁻¹² M, e.g, the association between thesynthetic regulator and the polypeptide has an affinity of from about10⁻³ M to about 5×10⁻³M, from about 5×10⁻³M to about 10⁻⁴ M, from about10⁻⁴ M to about 5×10⁻⁴M, from about 5×10⁻⁴ M to about 10⁻⁵ M, from about10⁻⁵ M to about 5×10⁻⁵ M, from about 5×10⁻⁵ M to about 10⁻⁶ M, fromabout 10⁻⁶ M to about 5×10⁻⁶M to about 10⁻⁷ M, from about 10⁻⁷ M toabout 5×10⁻⁷M, from about 5×10⁻⁷ M to about 10⁻⁸ M, from about 10⁻⁸ M toabout 5×10⁻⁴ M, from about 5×10⁻⁸ M to about 10⁻⁹M, from about 10⁻⁹ M toabout 5×10⁻⁹ M, from about 5×10⁻⁹ M to about 10⁻¹⁰ M, from about 10⁻¹⁰ Mto about 5×10⁻¹⁰ M, from about 5×10⁻¹⁰ M to about 10⁻¹¹ M, from about5×10⁻¹¹ M to about 10⁻¹² M, or greater. In some embodiments, e.g., wherea subject synthetic regulator comprises two or more polypeptideassociation moieties, each of the moieties can provide for attachment toa polypeptide with an affinity of less than about 10⁻⁹ M, but togetherthe two or more polypeptide association moieties provide for a bindingaffinity that is 10⁻⁹ M or greater. In some embodiments, e.g., where asubject synthetic regulator comprises two or more polypeptideassociation moieties, each of the moieties can provide for attachment toa polypeptide with an affinity of less than about 10⁻⁴ M, but togetherthe two or more polypeptide association moieties provide for a bindingaffinity that is 10⁻⁴M or greater.

Occlusion of a pore by a protein association moiety can involvephysically hindering access to a pore from the inside of the pore orfrom the outside of the pore. In some embodiments, the proteinassociation moiety is a blocker (e.g., a pore blocker of an ion channel,or an interaction domain that binds to other biological macromoleculessuch as polypeptides or nucleic acids). In some embodiments, the proteinassociation moiety can provide occlusion to pores of diameters of about1 angstrom, 2 angstroms, 3 angstroms, or 4 angstroms. In someembodiments, the protein association moiety can provide occlusion topores of diameters of about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 angstroms.

In certain embodiments, the polypeptide association moiety comprises agroup selected from hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀ alkyl,—NR¹⁰R¹¹, —NR¹²C(O)R¹³, C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, substituted C₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl,heteroaryl, heterocyclic, heterocyclooxy, heterocyclothio,heteroarylamino, heterocycloamino, C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀cycloalkyl, C₄₋₁₀ cycloalkenyl, substituted C₄₋₁₀ cycloalkenyl, cyano,halo, —OR¹⁰, —C(O)OR¹⁰, —SR¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰; wherein

R¹⁰ and R¹¹ are independently selected from hydrogen and C₁₋₁₀ alkyl;

R¹² is hydrogen or C₁₋₁₀ alkyl;

R¹³ is selected from hydrogen, C₁₋₁₀ alkyl, C₁₋₈ alkenyl, C₆₋₁₀ aryl,and substituted C₁₋₁₀ alkyl.

In certain embodiments, the polypeptide association moiety comprises agroup selected from hydrogen, alkyl, amino, substituted amino, andaminoacyl.

In certain embodiments, the polypeptide association moiety comprises agroup selected from hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀ alkyl,—NR¹⁰R¹¹, —NR¹²C(O)R¹³, C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, substituted C₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl,heteroaryl, heterocyclic, heterocyclooxy, heterocyclothio,heteroarylamino, heterocycloamino, C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀cycloalkyl, C₄₋₁₀ cycloalkenyl, substituted C₄₋₁₀ cycloalkenyl, cyano,halo, —OR¹⁰, —C(O)OR¹⁰, —SR¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰; wherein

R¹⁰ and R¹¹ are independently selected from hydrogen, C₁-C₁₀ alkyl,substituted C₁-C₁₀ alkyl, C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl,C₂₋₁₀ alkynyl, substituted C₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆-20aryl, C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀cycloalkenyl, and substituted C₄₋₁₀ cycloalkenyl;

R¹² is selected from hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substitutedC₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl, C₄₋₁₀ cycloalkyl,substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl, and substituted C₄₋₁₀cycloalkenyl; and

R¹³ is selected from hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substitutedC₂₋₁₀ alkynyl, C₆-C₁₀ aryl, substituted C₆₋₂₀ aryl, C₄₋₁₀ cycloalkyl,substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl, substituted C₄₋₁₀cycloalkenyl, CH₂—N(CH₂CH₃)₃ ⁺, and —CH₂—SO₃ ⁻.

Exemplary suitable polypeptide association moieties are depicted in FIG.4. In FIG. 4, polypeptide association moieties are designated “R.”

Photoisomerizable Group

The photoisomerizable group is one that changes from a first isomericform to a second isomeric form upon exposure to light of differentwavelengths, or upon a change in exposure from dark to light, or fromlight to dark. For example, in some embodiments, the photoisomerizablegroup is in a first isomeric form when exposed to light of a firstwavelength, and is in a second isomeric form when exposed to light of asecond wavelength. Suitable photoisomerizable groups includestereoisomers and constitutional isomers.

The first wavelength and the second wavelength can differ from oneanother by from about 1 nm to about 2000 nm or more, e.g., from about 1nm to about 10 nm, from about 10 nm to about 20 nm, from about 20 nm toabout 50 nm, from about 50 nm to about 75 nm, from about 75 nm to about100 nm, from about 100 nm to about 125 nm, from about 125 nm to about150 nm, or from about 150 nm to about 200 nm, from about 200 nm to about500 nm, from about 500 nm to about 800 nm, from about 800 nm to about1000 nm, from about 1000 nm to about 1500 nm, from about 1500 nm toabout 2000 nm, or more than 2000 nm.

In other embodiments, the photoisomerizable group is in a first isomericform when exposed to light of a wavelength λ₁, and is in a secondisomeric form in the absence of light (e.g., in the absence of light,the photoisomerizable group undergoes spontaneous relaxation into thesecond isomeric form). In these embodiments, the first isomeric form isinduced by exposure to light of wavelength λ₁, and the second isomericform is induced by not exposing the photoisomerizable group to light,e.g., keeping the photoisomerizable group in darkness. In otherembodiments, the photoisomerizable group is in a first isomeric form inthe absence of light, e.g., when the photoisomerizable group is in thedark; and the photoisomerizable group is in a second isomeric form whenexposed to light of a wavelength λ₁. In other embodiments, thephotoisomerizable group is in a first isomeric form when exposed tolight of a first wavelength λ₁, and the photoisomerizable group is in asecond isomeric form when exposed to light of second wavelength λ₂.

For example, in some embodiments, the photoisomerizable group is in atrans configuration in the absence of light, or when exposed to light ofa first wavelength; and the photoisomerizable group is in a cisconfiguration when exposed to light, or when exposed to light of asecond wavelength that is different from the first wavelength. Asanother example, in some embodiments, the photoisomerizable group is ina cis configuration in the absence of light, or when exposed to light ofa first wavelength; and the photoisomerizable group is in a transconfiguration when exposed to light, or when exposed to light of asecond wavelength that is different from the first wavelength.

The wavelength of light that effects a change from a first isomeric formto a second isomeric form ranges from 10⁻⁸ m to about 1 m, e.g., fromabout 10⁻⁸ m to about 10⁻⁷ m, from about 10⁻⁷ m to about 10⁻⁶ in, fromabout 10⁻⁶ m to about 10⁻⁴ m, from about 10⁻⁴ in to about 10⁻² m, orfrom about 10⁻² m to about 1 m. “Light,” as used herein, refers toelectromagnetic radiation, including, but not limited to, ultravioletlight, visible light, infrared, and microwave.

The wavelength of light that effects a change from a first isomeric formto a second isomeric form ranges in some embodiments from about 200 nmto about 800 nm, e.g., from about 200 nm to about 250 nm, from about 250nm to about 300 nm, from about 300 nm to about 350 nm, from about 350 nmto about 400 nm, from about 400 nm to about 450 nm, from about 450 nm toabout 500 nm, from about 500 nm to about 550 nm, from about 550 nm toabout 600 nm, from about 600 nm to about 650 nm, from about 650 nm toabout 700 nm, from about 700 nm to about 750 nm, or from about 750 nm toabout 800 nm, or greater than 800 nm.

In other embodiments, the wavelength of light that effects a change froma first isomeric form to a second isomeric form ranges from about 800 nmto about 2500 nm, e.g., from about 800 nm to about 900 nm, from about900 nm to about 1000 nm, from about 1000 nm to about 1200 nm, from about1200 nm to about 1400 nm, from about 1400 nm to about 1600 nm, fromabout 1600 nm to about 1800 nm, from about 1800 nm to about 2000 nm,from about 2000 nm to about 2250 nm, or from about 2250 nm to about 2500nm. In other embodiments, the wavelength of light that effects a changefrom a first isomeric form to a second isomeric form ranges from about 2nm to about 200 nm, e.g., from about 2 nm to about 5 nm, from about 5 nmto about 10 nm, from about 10 nm to about 25 nm, from about 25 nm toabout 50 nm, from about 50 nm to about 75 nm, from about 100 nm, fromabout 100 nm to about 150 nm, or from about 150 nm to about 200 nm.

The difference between the first wavelength and the second wavelengthcan range from about 1 nm to about 2000 nm or more, as described above.Of course, where the synthetic light regulator is switched from darknessto light, the difference in wavelength is from essentially zero to asecond wavelength.

The intensity of the light can vary from about 1 W/m² to about 50 W/m²,e.g., from about 1 W/m² to about 5 W/m², from about 5 W/m² to about 10W/m², from about 10 W/m², from about 10 W/m² to about 15 W/m², fromabout 15 W/m² to about 20 W/m², from about 20 W/m² to about 30 W/m²,from about 30 W/m² to about 40 W/m², or from about 40 W/m² to about 50W/m². The intensity of the light can vary from about 1 μW/cm² to about100 μW/cm², e.g., from about 1 μW/cm² to about 5 μW/cm², from about 5μW/cm² to about 10 μW/cm², from about 10 μW/cm² to about 20 μW/cm², fromabout 20 μW/cm² to about 25 μW/cm², from about 25 μW/cm² to about 50μW/cm², from about 50 μW/cm² to about 75 μW/cm², or from about 75 μW/cm²to about 100 μW/cm². In some embodiments, the intensity of light variesfrom about 1 μW/mm² to about 1 W/mm², e.g., from about 1 μW/mm² to about50 μW/mm², from about 50 μW/mm² to about 100 μW/mm², from about 100μW/mm² to about 500 μW/mm², from about 500 μW/mm² to about 1 mW/mm²,from about 1 mW/mm² to about 250 mW/mm², from about 250 mW/mm² to about500 mW/mm², or from about 500 mW/mm² to about 1 W/mm².

In some embodiments, the change from a first isomeric form to a secondisomeric form of the photoisomerizable group is effected using sound,instead of electromagnetic (EM) radiation (light). For example, in someembodiments, the change from a first isomeric form to a second isomericform of the photoisomerizable group is effected using ultrasound.

Photoisomerizable groups are known in the art, and any knownphotoisomerizable group can be included in a subject synthetic regulatorof protein function. Suitable photoisomerizable groups include, but arenot limited to, azobenzene and derivatives thereof; spiropyran andderivatives thereof; triphenyl methane and derivatives thereof;4,5-epoxy-2-cyclopentene and derivatives thereof; fulgide andderivatives thereof; thioindigo and derivatives thereof; diaryletheneand derivatives thereof; diallylethene and derivatives thereof;overcrowded alkenes and derivatives thereof; and anthracene andderivatives thereof. In some embodiments, a suitable photoisomerizablegroup is a photoisomerizable group as shown in the examples herein.

Suitable spiropyran derivatives include, but are not limited to,1,3,3-trimethylindolinobenzopyrylospiran;1,3,3-trimethylindolino-6′-nitrobenzopyrylospiran;1,3,3-trimethylinclolino-6′-bromobenzopyrylospiran;1-n-decyl-3,3-dimethylindolino-6′-nitrobenzopyrylospiran;1-n-octadecy-1-3,3-dimethylindolino-6′-nitrobenzopyrylospiran;3′,3′-dimethyl-6-nitro-1′-[2-(phenylcarbamoyl)ethyl]spiro;[2H-1-benzopyran-2,2′-indoline];1,3,3-trimethylindolino-8′-methoxybenzopyrylospiran; and1,3,3-trimethylindolino-β-naphthopyrylospiran. Also suitable for use isa merocyanine form corresponding to spiropyran or a spiropyranderivative.

Suitable triphenylmethane derivatives include, but are not limited to,malachite green derivatives, specifically, there can be mentioned, forexample, bis[dimethylamino)phenyl]phenylmethanol,bis[4-(diethylamino)phenyl]phenylmethanol,bis[4-(dibutylamino)phenyl]phenylmethanol andbis[4-(diethylamino)phenyl]phenylmethane.

Suitable 4,5-epoxy-2-cyclopentene derivatives include, for example,2,3-diphenyl-1-indenone oxide and 2′,3′-dimethyl-2,3-diphenyl-1-indenoneoxide.

Suitable azobenzene compounds include, e.g., compounds having azobenzeneresidues crosslinked to a side chain, e.g., compounds in which4-carboxyazobenzene is ester bonded to the hydroxyl group of polyvinylalcohol or 4-carboxyazobenzene is amide bonded to the amino group ofpolyallylamine. Also suitable are azobenzene compounds having azobenzeneresidues in the main chain, for example, those formed by ester bondingbis(4-hydroxyphenyl)dimethylmethane (also referred to as bisphenol A)and 4,4′-dicarboxyazobenzene or by ester bonding ethylene glycol and4,4′-dicarboxyazobenzene.

Suitable fulgide derivatives include, but are not limited to,isopropylidene fulgide and adamantylidene fulgide.

Suitable diallylethene derivatives include, for example,1,2-dicyano-1,2-bis(2,3,5-trimethyl-4-thienyl)ethane;2,3-bis(2,3,5-trimethyl-4-thiethyl) maleic anhydride;1,2-dicyano-1,2-bis(2,3,5-trimethyl-4-selenyl)ethane;2,3-bis(2,3,5-trimethyl-4-selenyl) maleic anhydride; and1,2-dicyano-1,2-bis(2-methyl-3-N-methylindole)ethane.

Suitable diarylethene derivatives include but are not limited to,substituted perfluorocyclopentene-bis-3-thienyls andbis-3-thienylmaleimides.

Suitable overcrowded alkenes include, but are not limited to,cis-2-nitro-7-(dimethylamino)-9-(2′,3′-dihydro-1′H-naphtho[2,1-b]thiopyran-1′-ylidene)-9H-thioxantheneandtrans-dimethyl-[1-(2-nitro-thioxanthen-9-ylidene)-2,3-dihydro-1H-benzo[f]thiochromen-8-yl]amine.Overcrowded alkenes are described in the literature. See, e.g., terWielet al. (2005) Org. Biomol. Chem. 3:28-30; and Geertsema et al. (1999)Agnew CHem. Int. Ed. Engl. 38:2738.

Other suitable photoisomerizable moieties include, e.g., reactive groupscommonly used in affinity labeling, including diazoketones, aryl azides,diazerenes, and benzophenones.

Ligands

As used herein, the term “ligand” refers to a molecule (e.g., a smallmolecule, a peptide, or a protein) that binds to a polypeptide andeffects a change in an activity of the polypeptide, and/or effects achange in conformation of the polypeptide, and/or affects binding ofanother polypeptide to the polypeptide. Ligands include agonists,partial agonists, inverse agonists, antagonists, allosteric modulators,and blockers.

In some embodiments, the ligand is a naturally-occurring ligand. Inother embodiments, the ligand is a synthetic ligand. In otherembodiments, the ligand is an endogenous ligand. In some embodiments,the ligand is an agonist. In other embodiments, the ligand is an inverseagonist. In other embodiments, the ligand is a partial agonist. In otherembodiments, the ligand is an antagonist. In other embodiments, theligand is an allosteric modulator. In other embodiments, the ligand is ablocker. The term “antagonist” generally refers to an agent that bindsto a ligand-binding polypeptide and inhibits an activity of theligand-binding polypeptide. An “antagonist” may be an agent that bindsto an allosteric site but does not activate the ligand-bindingpolypeptide; instead, the antagonist generally excludes binding by anagonist and thus prevents or hinders activation. The term “blocker”refers to an agent that acts directly on the active site, pore, orallosteric site. Ligands suitable for use herein bind reversibly to aligand-binding site of a ligand-binding polypeptide.

The ligand is selected based in part on the activity of the polypeptideto which the synthetic regulator will be attached. For example, a ligandfor a hormone-binding transcription factor is a hormone, or a syntheticanalog of the hormone. A ligand for a tetracycline transactivator istetracycline or a synthetic analog thereof. A ligand for an enzyme willin some embodiments be a synthetic agonist or antagonist of the enzyme.In some embodiments, a ligand will block the ligand-binding site. Aligand for a ligand-gated ion channel will in some embodiments be anaturally-occurring ligand, or a synthetic version of the ligand, e.g.,a synthetic analog of the ligand. In some embodiments, the ligand isother than an acetylcholine receptor ligand. In some embodiments, theligand is other than trimethylammonium.

In some embodiments, a ligand is a small molecule ligand. Small moleculeligands generally have a molecular weight in a range of from about 50daltons to about 3000 daltons, e.g., from about 50 daltons to about 75daltons, from about 75 daltons to about 100 daltons, from about 100daltons to about 250 daltons, from about 250 daltons to about 500daltons, from about 500 daltons to about 750 daltons, from about 750daltons to about 1000 daltons, from about 1000 daltons to about 1250daltons, from about 1250 daltons to about 1500 daltons, from about 1500daltons to about 2000 daltons, from about 2000 daltons to about 2500daltons, or from about 2500 daltons to about 3000 daltons.

In other embodiments, a ligand is a peptide ligand. Peptide ligands canhave a molecular weight in a range of from about 1 kDa to about 20 kDa,e.g., from about 1 kDa to about 2 kDa, from about 2 kDa to about 5 kDa,from about 5 kDa to about 7 kDa, from about 7 kDa to about 10 kDa, fromabout 10 kDa to about 12 kDa, from about 12 kDa to about 15 kDa, or fromabout 15 kDa to about 20 kDa.

Suitable ligands include, but are not limited to, ligands that block oractivate the function of a ligand-binding protein, where ligand-bindingproteins include channels; receptors (including, but not limited to,ionotropic receptors that bind transmitters; ionotropic receptors thatbind hormones; metabotropic receptors; receptor tyrosine kinases; growthfactor receptors; and other membrane receptors that signal by binding tosoluble or membrane-bound or extracellular matrix-bound small moleculesor proteins); transporters (including but not limited to iontransporters, organic molecule transporters, peptide transporters, andprotein transporters); enzymes (including but not limited to kinases;phosphatases; ubiquitin ligases; acetylases; oxo-reductases; lipases;enzymes that add lipid moieties to proteins or remove them; proteases;and enzymes that modify nucleic acids, including but not limited toligases, helicases, topoisomerases, and telomerases); motor proteins(including kinesins, dyenins and other microtobule-based motors, myosinsand other actin-based motors, DNA and RNA polymerases and other motorsthat track along polynucleotides); scaffolding proteins; adaptorproteins; cytoskeletal proteins; and other proteins that localize ororganize protein domains and superstructures within cells.

Suitable ligands include, but are not limited to, ligands that functionas general anesthetics; ligands that function as local anesthetics;ligands that function as analgesics; synthetic and semi-synthetic opioidanalgesics (e.g., phenanthrenes, phenylheptylamines, phenylpiperidines,morphinans, and benzomorphans) where exemplary opioid analgesics includemorphine, oxycodone, fentanyl, pentazocine, hydromorphone, meperidine,methadone, levorphanol, oxymorphone, levallorphan, codeine,dihydrocodeine, hydrocodone, propoxyphene, nalmefene, nalorphine,naloxone, naltrexone, buprenorphine, butorphanol, nalbuphine, andpentazocine; ionotropic glutamate receptor agonists and antagonists,e.g., N-methyl-D-aspartate (NMDA) receptor agonists and antagonists,kainate (KA) receptor agonists and antagonists, andα-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptoragonists and antagonists; non-opioid analgesics, e.g., acetylsalicylicacid, choline magnesium trisalicylate, acetaminophen, ibuprofen,fenoprofen, diflusinal, and naproxen; muscarinic receptor agonists;muscarinic receptor antagonists; acetylcholine receptor agonists;acetylcholine receptor antagonists; serotonin receptor agonists;serotonin receptor antagonists; enzyme inhibitors; a benzodiazepine,e.g. chlordiazepoxide, clorazepate, diazepam, flurazepam, lorazepam,oxazepam, temazepam or triazolam; a barbiturate sedative, e.g.amobarbital, aprobarbital, butabarbital, butabital, mephobarbital,metharbital, methohexital, pentobarbital, phenobartital, secobarbital,talbutal, theamylal, or thiopental; an H₁ antagonist having a sedativeaction, e.g. diphenhydramine, pyrilamine, promethazine,chlorpheniramine, or chlorcyclizine; an NMDA receptor antagonist, e.g.dextromethorphan ((+)-3-hydroxy-N-methylmorphinan) or its metabolitedextrorphan ((+)-3-hydroxy-N-methylmorphinan), ketamine, memantine,pyrroloquinoline quinine, cis-4-(phosphonomethyl)-2-piperidinecarboxylicacid, budipine, topiramate, neramexane, or perzinfotel; analpha-adrenergic, e.g. doxazosin, tamsulosin, clonidine, guanfacine,dexmetatomidine, modafinil, phentolamine, terazasin, prazasin or4-amino-6,7-dimethoxy-2-(5-methane-sulfonamido-1,2,3,4-tetrahydroisoquinol-2-yl)-5-(2-pyridyl)quinazoline;a tricyclic antidepressant, e.g. desipramine, imipramine, amitriptyline,or nortriptyline; an anticonvulsant, e.g. carbamazepine, lamotrigine,topiratmate, or valproate; a tachykinin (NK) antagonist, particularly anNK-3, NK-2 or NK-1 antagonist, e.g.(α-R,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-tetrahydro-9-methyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g][1,7]-naphthyridine-6-13-dione(TAK-637),5-[[(2R,3S)-2-[(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy-3-(4-fluorophenyl)-4-morpholinyl]-methyl]-1,2-dihydro-3H-1,2,4-triazol-3-one(MK-869), aprepitant, lanepitant, dapitant or3-[[2-methoxy-5-(trifluoromethoxy)phenyl]-methylamino]-2-phenylpiperidine(2S,3S); a muscarinic antagonist, e.g oxybutynin, tolterodine,propiverine, tropsium chloride, darifenacin, solifenacin, temiverine, oripratropium; a cyclooxygenase-2 (COX-2) selective inhibitor, e.g.celecoxib, rofecoxib, parecoxib, valdecoxib, deracoxib, etoricoxib, orlumiracoxib; a vanilloid receptor agonist (e.g. resinferatoxin) orantagonist (e.g. capsazepine); a beta-adrenergic such as propranolol; a5-HT receptor agonist or antagonist, e.g., a 5-HT₁B/₁D agonist such aseletriptan, sumatriptan, naratriptan, zolmitriptan or rizatriptan; a5-HT₂A receptor antagonist such asR(+)-α-(2,3-dimethoxy-phenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol(MDL-100907); and the like.

Suitable ligands for Na⁺ channels include, but are not limited to,lidocaine, novocaine, xylocaine, lignocaine, novocaine, carbocaine,etidocaine, procaine, prontocaine, prilocalnc, bupivacaine, cinchocaine,mepivacaine, quinidine, flecamide, procaine,N-[[2′-(aminosulfonyl)biphenyl-4-yl]methyl]-N′-(2,2′-bithien-5-ylmethyl)succinamide(BPBTS), QX-314, saxitoxin, tetrodotoxin, and a type III conotoxin.Suitable ligands for Na⁺ channels also include, but are not limited to,tetrodotoxin, saxitoxin, guanidinium, polyamines (e.g. spermine,cadaverine, putrescine, μ-conotoxin, and δ-conotoxin.

Suitable ligands for K⁺ channels include, but are not limited to,quaternary ammonium (e.g., tetraethyl ammonium, tetrabutylammonium,tetrapentylammonium), 4-aminopyridine, sulfonylurea, Glibenclamide;Tolbutamide; Phentolamine, qiunine, qunidine, peptide toxins (e.g.,charybdotoxin, agitoxin-2, apamin, dendrotoxin, VSTX1, hanatoxin-1,hanatoxin-2, and Tityus toxin K-α.

Suitable ligands for CNG and HCN channels include, but are not limitedto, 1-cis diltiazem and ZD7288. Suitable ligands for glycine receptorsinclude, but are not limited to, strychnine and picrotoxin.

Suitable ligands for nicotinic acetylcholine receptors include, but arenot limited to, (+)tubocurarine, Methyllycaconitine, gallamine,Nicotine; Anatox in A, epibatidine, ABT-94, Lophotoxin, Cytisine,Hexamethonium, Mecamylamine, and Dihydro-β-erythroidine. Suitableligands for muscarinic acetylcholine receptors include, but are notlimited to, a muscarinic acetylcholine receptor antagonist as describedin U.S. Pat. No. 7,439,255; AF267B (see, e.g., U.S. Pat. No. 7,439,251);phenylpropargyloxy-1,2,5-thiadiazole-quinuclidine; carbachol;pirenzapine; migrastatin; a compound as described in U.S. Pat. No.7,232,841; etc.

Suitable ligands for GABA receptors include, but are not limited to,Muscimol, TIIIP, Procabide, bicuculine, picrotoxin, gabazine,gabapentin, diazepam, clonazepam, flumazenil, a β-carboline carboxylateethyl ester, baclofen, faclofen, and a barbiturate.

Many suitable ligands will be known to those skilled in the art; and thechoice of ligand will depend, in part, on the target (e.g., receptor,ion channel, enzyme, etc.) to which the ligand binds.

Exemplary Synthetic Regulators

In some embodiments, a subject synthetic regulator is a compound havingthe formula: (A)_(n)-X₁—(B)_(m)—X₂—(C)_(p), where:

A is a polypeptide association moiety that comprises a group selectedfrom hydrogen, C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, —NR¹⁰R¹¹,—NR¹²C(O)R¹³, C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,substituted C₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl,heteroaryl, heterocyclic, heterocyclooxy, heterocyclothio,heteroarylamino, heterocycloamino, C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀cycloalkyl, C₄₋₁₀ cycloalkenyl, substituted C₄₋₁₀ cycloalkenyl, cyano,halo, —OR¹⁰, —C(O)OR¹⁰, —SR¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰; where R¹⁰ and R¹¹are independently selected from hydrogen and C₁₋₁₀ alkyl; R¹² ishydrogen or C₁₋₁₀ alkyl; R¹³ is selected from hydrogen, C₁₋₁₀ alkyl,C₁₋₈ alkenyl, C₆₋₁₀ aryl, and substituted C₁₋₁₀ alkyl;

B is a photoisomerizable group;

C is a ligand;

each of n, m, and p is independently an integer from 1 to 10;

X₁, when present, is a spacer; and

X₂, when present, is a spacer.

Suitable ligands include those described above. In some of theseembodiments, the ligand is a sodium channel ligand, a synthetic ligand,a ligand that binds to a ligand binding site of an ionotropic receptor,a ligand that hinds to a ligand binding site of a metabotropic receptor,a ligand that functions as an anesthetic, a potassium channel ligand, agamma aminobutyric acid receptor ligand. In some of these embodiments,the ligand is a sodium channel ligand, a potassium channel ligand, or agamma aminobutyric acid receptor ligand. In some of these embodiments,the ligand is an agonist, an antagonist, an allosteric modulator, or ablocker.

In some embodiments, a subject synthetic regulator is a compound havingthe formula: (A)_(n)-X₁—(B)_(m)—X₂—(C)_(p), where:

A is a polypeptide association moiety that comprises a group selectedfrom hydrogen, C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, —NR¹⁰R¹¹,—NR¹²C(O)R¹³, C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,substituted C₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl,heteroaryl, heterocyclic, heterocyclooxy, heterocyclothio,heteroarylamino, heterocycloamino, C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀cycloalkyl, C₄₋₁₀ cycloalkenyl, substituted C₄₋₁₀ cycloalkenyl, cyano,halo, —OR¹⁰, —C(O)OR¹⁰, SR¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰; where R¹⁰ and R¹¹ areindependently selected from hydrogen and C₁₋₁₀ alkyl; R¹² is hydrogen orC₁₋₁₀ alkyl; R¹³ is selected from hydrogen, C₁₋₁₀ alkyl, C₁₋₈ alkenyl,C₆₋₁₀ aryl, and substituted C₁₋₁₀ alkyl;

B is a photoisomerizable group selected from an azobenzene, a fulgide, aspiropyran, a triphenyl methane, a thioindigo, a diarylethene, or anovercrowded alkene;

C is a ligand;

each of n, m, and p is independently an integer from 1 to 10;

X₁, when present, is a spacer; and

X₂, when present, is a spacer.

Suitable ligands include those described above. In some of theseembodiments, the ligand is a sodium channel ligand, a synthetic ligand,a ligand that binds to a ligand binding site of an ionotropic receptor,a ligand that binds to a ligand binding site of a metabotropic receptor,a ligand that functions as an anesthetic, a potassium channel ligand, agamma aminobutyric acid receptor ligand. In some of these embodiments,the ligand is a sodium channel ligand, a potassium channel ligand, or agamma aminobutyric acid receptor ligand. In some of these embodiments,the ligand is an agonist, an antagonist, an allosteric modulator, or ablocker.

In some embodiments, a subject synthetic regulator is a compound havingthe formula: (A)_(n)-X₁—(B)_(m)—X₂—(C)_(p), where:

A is a polypeptide association moiety that comprises a group selectedfrom hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀ alkyl, —NR¹⁰R¹¹,—NR¹²C(O)R¹³, C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,substituted C₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl,heteroaryl, heterocyclic, heterocyclooxy, heterocyclothio,heteroarylamino, heterocycloamino, C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀cycloalkyl, C₄₋₁₀ cycloalkenyl, substituted C₄₋₁₀ cycloalkenyl, cyano,halo, —OR¹⁰, —C(O)OR¹⁰, —SR¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰; where R¹⁰ and R¹¹are independently selected from hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,substituted C₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl, C₄₋₁₀cycloalkyl, substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl, andsubstituted C₄₋₁₀ cycloalkenyl; R¹² is selected from hydrogen, C₁₋₁₀alkyl, substituted C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, substituted C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, substituted C₂₋₁₀ alkynyl, C₆₋₂₀ aryl,substituted C₆₋₂₀ aryl, C₄₋₁₀cycloalkyl, C₄₋₁₀ cycloalkyl, substitutedC₄₋₁₀ cycloalkyenyl, and substituted C₄₋₁₀ cycloalkenyl; and R¹³ isselected from hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀ alkyl. C₂₋₁₀alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substituted C₂₋₁₀alkenyl, C₆-C₁₀ aryl, substituted C₆₋₂₀ aryl, C₄₋₁₀ cycloalkyl,substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl, substituted C₄₋₁₀cycloalkenyl, CH₂—N(CH₂CH₃)₃ ⁺, and —CH₂—SO₃ ⁻;

B is a photoisomerizable group;

C is a ligand;

each of n, m, and p is independently an integer from 1 to 10;

X₁, when present, is a spacer; and

X₂, when present, is a spacer.

Suitable ligands include those described above. In some of theseembodiments, the ligand is a sodium channel ligand, a synthetic ligand,a ligand that binds to a ligand binding site of an ionotropic receptor,a ligand that binds to a ligand binding site of a metabotropic receptor,a ligand that functions as an anesthetic, a potassium channel ligand, agamma aminobutyric acid receptor ligand. In some of these embodiments,the ligand is a sodium channel ligand, a potassium channel ligand, or agamma aminobutyric acid receptor ligand. In some of these embodiments,the ligand is an agonist, an antagonist, an allosteric modulator, or ablocker.

In some embodiments, a subject synthetic regulator is a compound havingthe formula: (A)_(n)-X₁—(B)_(m)—X₂—(C)_(p), wherein:

A is a polypeptide association moiety that comprises a group selectedfrom hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀ alkyl, —NR¹⁰R¹¹,—NR¹²C(O)R¹³, C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,substituted C₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl,heteroaryl, heterocyclic, heterocyclooxy, heterocyclothio,heteroarylamino, heterocycloamino, C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀cycloalkyl, C₄₋₁₀ cycloalkenyl, substituted C₄₋₁₀ cycloalkenyl, cyano,halo, —OR¹⁰, —C(O)OR¹⁰, —SR¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰; where R¹⁰ and R¹¹are independently selected from hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,substituted C₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl, C₄₋₁₀cycloalkyl, substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl, andsubstituted C₄₋₁₀ cycloalkenyl; R¹² is selected from hydrogen, C₁₋₁₀alkyl, substituted C₁₋₁₄ alkyl, C₂₋₁₀ alkenyl, substituted C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, substituted C₂₋₁₀ alkynyl, C₅₋₂₀ aryl,substituted C₆₋₂₀ aryl, C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀ cycloalkyl,C₄₋₁₀ cycloalkenyl, and substituted C₄₋₁₀ cycloalkenyl; and R¹³ isselected from hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substituted C₂₋₁₀alkynyl, C₆-C₁₀ aryl, substituted C₆₋₂₀ aryl, C₄₋₁₀ cycloalkyl,substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl, substituted C₄₋₁₀cycloalkenyl, CH₂—N(CH₂CH₃)₃ ⁺, and —CH₂—SO₃ ⁻;

B is a photoisomerizable group selected from an azobenzene, a fulgide, aspiropyran, a triphenyl methane, a thioindigo, a diarylethene, or anovercrowded alkene;

C is a ligand;

each of n, m, and p is independently an integer from 1 to 10;

X₁, when present, is a spacer; and

X₂, when present, is a spacer.

Suitable ligands include those described above. In some of theseembodiments, the ligand is a sodium channel ligand, a synthetic ligand,a ligand that binds to a ligand binding site of an ionotropic receptor,a ligand that hinds to a ligand binding site of a metabotropic receptor,a ligand that functions as an anesthetic, a potassium channel ligand, agamma aminobutyric acid receptor ligand. In some of these embodiments,the ligand is a sodium channel ligand, a potassium channel ligand, or agamma aminobutyric acid receptor ligand. In some of these embodiments,the ligand is an agonist, an antagonist, an allosteric modulator, or ablocker.

In certain embodiments, a subject synthetic regulator functions as ablocker (e.g., a potassium channel blocker, and/or a sodium channelblocker and/or a calcium channel blocker) in the cis-isomeric form. Inother embodiments, a subject synthetic regulator functions as a blocker(e.g., a potassium channel blocker, and/or a sodium channel blockerand/or a calcium channel blocker) in the trans-isomeric form.

In some embodiments, a subject synthetic regulator of polypeptidefunction is a compound of Formula XI:

wherein Q¹ is CH₂— or —C(═O)—;

Q² is

each of R¹ are independently selected from hydrogen, C₁₋₁₀ alkyl,substituted C₁₋₁₀ alkyl, —NR¹⁰R¹¹, —NR¹²C(O)R¹³, C₂₋₁₀ alkenyl,substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substituted C₂₋₁₀ alkynyl,C₆₋₂₀ aryl, substituted C₅₋₂₀ aryl, heteroaryl, heterocyclic,heterocyclooxy, heterocyclothio, heteroarylamino, heterocycloamino,C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl,substituted C₄₋₁₀ cycloalkenyl, cyano, halo, —OR¹⁰, —C(O)OR¹⁰, —S(O)R¹⁰,—S(O)₂R¹⁰;

x is an integer from 1 to 5;

y is an integer from 1 to 4;

R² is selected from hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substitutedC₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl, C₄₋₁₀ cycloalkyl,substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl, and substituted C₄₋₁₀cycloalkenyl;

R³, R⁴, and R⁵ are independently selected from hydrogen, C₂-C₁ alkyl,substituted C₂-C₁₀ alkyl, C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl,C₂₋₁₀ alkynyl, substituted C₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆-20aryl, C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀cycloalkenyl, and substituted C₄₋₁₀ cycloalkenyl;

each of R⁶ are independently selected from hydrogen, C₁₋₁₀ alkyl,substituted C₁₋₁₀ alkyl, —NR¹⁰R¹¹, —NR¹²C(O)R¹³, C₂₋₁₀ alkenyl,substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substituted C₂₋₁₀ alkynyl,C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl, heteroaryl, heterocyclic,heterocyclooxy, heterocyclothio, heteroarylamino, heterocycloamino,C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl,substituted C₄₋₁₀ cycloalkenyl, cyano, halo, —OR¹⁰, —C(O)OR¹⁰, SR¹⁰,—S(O)R¹⁰, —S(O)₂R¹⁰;

R¹⁰ and R¹¹ are independently selected from hydrogen, C₁₋₁₀ alkyl,substituted C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, substituted C₂₋₁₀ substituted C₆₋₂₀ alkynyl, C₆₋₂₀ aryl, aryl,C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl, andsubstituted C₄₋₁₀ cycloalkenyl;

R¹² is selected from hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, substituted C₂₋₄₀ alkenyl, C₂₋₁₀ alkynyl, substitutedC₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl, C₄₋₁₀ cycloalkyl,substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl, and substituted C₄₋₁₀cycloalkenyl;

R¹³ is selected from hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substitutedC₂₋₁₀ alkynyl, C₆-C₁₀ aryl, substituted C₆₋₂₀ aryl, C₄₋₁₀ cycloalkyl,substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl, substituted C₄₋₁₀cycloalkenyl, —CH₂—N(CH₂CH₃)₃ ⁺, and —CH₂—SO₃ ⁻;

or a pharmaceutically acceptable salt thereof.

In some embodiments, a subject synthetic regulator of polypeptidefunction is a compound of Formula XII:

wherein

each of R¹ are independently selected from hydrogen, C₁₋₁₀ alkyl,substituted C₁₋₁₀ alkyl, —NR¹⁰R¹¹, NR¹²C(O)R¹³, C₂₋₁₀ alkenyl,substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substituted C₂₋₁₀ alkynyl,C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl, heteroaryl, heterocyclic,heterocyclooxy, heterocyclothio, heteroarylamino, heterocycloamino,C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl,substituted C₄₋₁₀ cycloalkenyl, cyano, halo, —OR¹⁰, —C(O)OR¹⁰, SR¹⁰,—S(O)R¹⁰, —S(O)₂R¹⁰;

x is an integer from 1 to 5;

y is an integer from 1 to 4;

R² is selected from hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substitutedC₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl, C₄₋₁₀ cycloalkyl,substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl, and substituted C₄₋₁₀cycloalkenyl;

R³, R⁴, and R⁵ are independently selected from hydrogen, C₂₋₈ alkyl,substituted C₂₋₁₀ alkyl, C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, substituted C₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl,C₄₋₁₀cycloalkyl, substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl, andsubstituted C₄₋₁₀ cycloalkenyl;

each of R⁶ are independently selected from hydrogen, C₁₋₁₀ alkyl,substituted C₁₋₁₀ alkyl, —NR¹⁰R¹¹, —NR¹²C(O)R¹³, C₂₋₁₀ alkenyl,substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substituted C₂₋₁₀ alkynyl,C₆₋₂ aryl, substituted C₆₋₂ aryl, heteroaryl, heterocyclic,heterocyclooxy, heterocyclothio, heteroarylamino, heterocycloamino,C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl,substituted C₄₋₁₀ cycloalkenyl, cyano, halo, —OR¹⁰, —C(O)OR¹⁰, —SR¹⁰,—S(O)R¹⁰, —S(O)₂R¹⁰;

R¹⁰ and R¹¹ are independently selected from hydrogen, C₁₋₁₀ alkyl,substituted C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, substituted C₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₅₋₂₀ aryl,C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl, andsubstituted C₄₋₁₀ cycloalkenyl;

R¹² is selected from hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substitutedC₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆₋₂₁) aryl, C₄₋₁₀ cycloalkyl,substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl, and substituted C₄₋₁₀cycloalkenyl;

R¹³ is selected from hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substitutedC₂₋₁₀ alkynyl, C₆-C₁₀ aryl, substituted C₆₋₂₀ aryl, C₄₋₁₀ cycloalkyl,substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl, substituted C₄₋₁₀cycloalkenyl, —CH₂—N(CH₂CH₃)₃ ⁺, and —CH₂—SO₃ ⁻;

or a pharmaceutically acceptable salt thereof.

In certain embodiments of Formula XL Q¹ is —CH₂—. In certain embodimentsof Formula XI, Q¹ is —C(═O)—.

In certain embodiments of Formula XI, Q² is

In certain embodiments of Formula XI, Q² is

In certain embodiments of any one of the above Formulae XI and XII, R³,R⁴, and R⁵ are C₂₋₁₀ alkyl. In certain embodiments of any one of theabove Formulae XI and XII, R³, R⁴, and R⁵ are C₂₋₅ alkyl. In certainembodiments of any one of the above Formulae XI and XII, R³, R⁴, and R⁵are C₂ alkyl. In certain embodiments of any one of the above Formulae XIand XII, R³, R⁴, and R⁵ are C₃ alkyl. In certain embodiments of any oneof the above Formulae XI and XII, R³, R⁴, and R⁵ are C₄ alkyl. Incertain embodiments of Formulae XI and XII, R³, R⁴, and R⁵ are hydrogen.

In certain embodiments of any one of the above Formulae XI and XII, R³,R⁴, and R⁵ are independently selected from C₂₋₈ alkyl or substitutedC₂₋₈ alkyl. In certain embodiments of any one of the above Formulae XIand XII, R³, R⁴, and R⁵ are independently selected from C₂₋₁₀ alkenyl,substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substituted C₂₋₁₀ alkynyl. Incertain embodiments of any one of the above Formulae XI and XII, R³, R⁴,and R⁵ are independently selected from C₆₋₂₀ aryl or substituted C₆₋₂₀aryl. In certain embodiments of any one of the above Formulae XI andXII, R³, R⁴, and R⁵ are independently selected from C₄₋₁₀ cycloalkyl,substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl, or substituted C₄₋₁₀cycloalkenyl.

In certain embodiments of any one of the above Formulae XI and XII, R²is hydrogen. In certain embodiments of any one of the above Formulae XIand XII, R² is C₁₋₁₀ alkyl. In certain embodiments of any one of theabove Formulae XI and XII, R² is C₁₋₅ alkyl. In certain embodiments ofany one of the above Formulae XI and XII, R² is hydrogen or C₁₋₅ alkyl.

In certain embodiments of any one of the above Formulae XI and XII, R²is C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl. In certain embodiments of anyone of the above Formulae XI and XII, R² is C₂₋₁₀ alkenyl, substitutedC₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substituted C₂₋₁₀ alkynyl. In certainembodiments of any one of the above Formulae XI and XII, R² is C₆₋₂₀aryl or substituted C₆₋₂₀ aryl. In certain embodiments of any one of theabove Formulae XI and XII, R² is C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀cycloalkyl, C₄₋₁₀ cycloalkenyl, or substituted C₄₋₁₀ cycloalkenyl.

In certain embodiments of any one of the above Formulae XI and XII, atleast one of R⁶ is C₁₋₁₀ alkyl, substituted C₁₋₁₀ alkyl, or halo. Incertain embodiments of any one of the above Formulae XI and XII, atleast one of R⁶ is C₁₋₄ alkyl. In certain embodiments of any one of theabove Formulae XI and XII, at least one of R⁶ is halo.

In certain embodiments of any one of the above Formulae XI and XII, atleast one of R⁶ is —NR¹⁰R¹¹ or —NR¹²C(O)R¹³. In certain embodiments ofany one of the above Formulae XI and XII, at least one of R⁶ is C₂₋₁₀alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, or substituted C₂₋₁₀alkynyl. In certain embodiments of any one of the above Formulae XI andXII, at least one of R⁶ is C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl,heteroaryl, or heterocyclic. In certain embodiments of any one of theabove Formulae XI and XII, at least one of R⁶ is heterocyclooxy,heterocyclothio, heteroarylamino, or heterocycloamino. In certainembodiments of any one of the above Formulae XI and XII, at least one ofR⁶ is C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀cycloalkenyl, or substituted C₄₋₁₀ cycloalkenyl. In certain embodimentsof any one of the above Formulae XI and XII, at least one of R⁶ iscyano, halo, —OR¹⁰, —C(O)OR¹⁰, —SR¹⁰, —S(O)R¹⁰, or —S(O)₂R¹⁰.

In certain embodiments of any one of the above Formulae XI and XII, atleast one of R¹ is hydrogen.

In certain embodiments of any one of the above Formulae XI and XII, atleast one of R¹ is C₁₋₈ alkyl, e.g., C₁₋₆ alkyl, C₁₋₅ alkyl or C₁₋₄alkyl. In some embodiments of any one of the above Formulae XI and XII,at least one of R¹ is C₁₋₄ alkyl.

In certain embodiments of any one of the above Formulae XI and XII, atleast one of R¹ is —NR¹²C(O)R¹³.

In certain embodiments of any one of the above Formulae XI and XII, atleast one of R¹ is —NR¹⁰R¹¹.

In certain embodiments of any one of the above Formulae XI and XII, atleast one of R¹ is C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl.

In certain embodiments of any one of the above Formulae XI and XII, atleast one of R¹ is C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, or substituted C₂₋₁₀ alkynyl. In certain embodiments of any oneof the above Formulae XI and XII, at least one of R¹ is C₆₋₂₀ aryl orsubstituted C₆₋₂₀ aryl. In certain embodiments of any one of the aboveFormulae XI and XII, at least one of R¹ is heteroaryl, heterocyclic,heterocyclooxy, heterocyclothio, heteroarylamino, or heterocycloamino.In certain embodiments of any one of the above Formulae XI and XII, atleast one of R¹ is C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀cycloalkenyl, or substituted C₄₋₁₀ cycloalkenyl. In certain embodimentsof any one of the above Formulae XI and XII, at least one of Fe iscyano, halo, —OR¹⁰, —C(O)OR¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰.

In certain embodiments of any one of the above Formulae XI and XII, R¹²is hydrogen. In certain embodiments of any one of the above Formulae XIand XII, R¹² is C₁₋₁₀ alkyl. In certain embodiments of any one of theabove Formulae XI and XII, R¹² is C₁₋₁₀ alkyl. In certain embodiments ofany one of the above Formulae XI and XII, R¹² is hydrogen or C₁₋₅ alkyl.

In certain embodiments of any one of the above Formulae XI and XII, R¹²is hydrogen. In certain embodiments of any one of the above Formulae XIand XII, R¹² is C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl. In certainembodiments of any one of the above Formulae XI and XII, R¹² is C₂₋₁₀alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, or substituted C₂₋₁₀alkynyl. In certain embodiments of any one of the above Formulae XI andXII, R¹² is C₆₋₂₀ aryl or substituted C₆₋₂₀ aryl. In certain embodimentsof any one of the above Formulae XI and XII, R¹² is C₄₋₁₀ cycloalkyl,substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl, or substituted C₄₋₁₀cycloalkenyl.

In certain embodiments of any one of the above Formulae XI and XII, R¹³is hydrogen or C₁₋₁₀ alkyl. In certain embodiments of any one of theabove Formulae XI and XII, R¹³ is C₁₋₁₀ alkyl. In certain embodiments ofany one of the above Formulae XI and X₁₁, R¹³ is C₁₋₅ alkyl. In certainembodiments of any one of the above Formulae XI and XII, R¹³ is hydrogenor C₁₋₅ alkyl.

In certain embodiments of any one of the above Formulae XI and XII, R¹³is alkenyl or substituted alkenyl. In certain embodiments of any one ofthe above Formulae XI and XII, R¹³ is C₁₋₁₀ alkenyl. In certainembodiments of any one of the above Formulae XI and XII, R¹³ is C₁₋₅alkenyl. In certain embodiments of any one of the above Formulae XI andXII, R¹³ is hydrogen or C₁₋₅ alkenyl.

In certain embodiments of any one of the above Formulae XI and XII, R¹³is C₆ aryl or substituted C₆ aryl.

In certain embodiments of any one of the above Formulae XI and XII, R¹³is —CH₂—N(CH₂CH₃)₃ ⁺ or —CH₂—SO₃ ⁻. In certain embodiments of any one ofthe above Formulae XI and XII, R¹³ is —CH₂—N(CH₂CH₃)₃ ⁺. In certainembodiments of any one of the above Formulae XI and XII, R¹³ is or—CH₂—SO₃ ⁻.

In certain embodiments of any one of the above Formulae XI and XII, R¹³is hydrogen. In certain embodiments of any one of the above Formulae XIand XII, R¹³ is C₁₋₁₀ alkyl or substituted C₁₋₁₀ alkyl. In certainembodiments of any one of the above Formulae XI and XII, R¹³ is C₂₋₁₀alkenyl or substituted C₂₋₁₀ alkenyl. In certain embodiments of any oneof the above Formulae XI and XII, R¹³ is C₂₋₁₀ alkynyl or substitutedC₂₋₁₀ alkynyl. In certain embodiments of any one of the above FormulaeXI and XII, R¹³ is C₆₋₁₀ aryl or substituted C₆₋₂₀ aryl. In certainembodiments of any one of the above Formulae XI and XII, R¹³ is C₄₋₁₀cycloalkyl, substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl, orsubstituted C₄₋₁₀ cycloalkenyl.

In certain embodiments of any one of the above Formulae XI and XII, atleast one of R¹⁰ and R¹¹ is hydrogen. In certain embodiments of any oneof the above Formulae XI and XII, at least one of R¹⁰ and R¹¹ is C₁₋₁₀alkyl or substituted C₁₋₁₀ alkyl. In certain embodiments of any one ofthe above Formulae XI and XII, at least one of R¹⁰ and R¹¹ is C₂₋₁₀alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, or substituted C₂₋₁₀alkynyl. In certain embodiments of any one of the above Formulae XI andXII, at least one of R¹⁰ and R¹¹ is C₆₋₂₀ aryl or substituted C₆₋₂₀aryl. In certain embodiments of any one of the above Formulae XI andXII, at least one of R¹⁰ and R¹¹ is C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀cycloalkyl, C₄₋₁₀ cycloalkenyl, or substituted C₄₋₁₀ cycloalkenyl.

In certain embodiments of any one of the above Formulae XI and XII, atleast one of R¹⁰ and R¹¹ is C₁₋₁₀ alkyl. In certain embodiments of anyone of the above Formulae XI and XII, at least one of R¹⁰ and R¹¹ isC₂₋₅ alkyl. In certain embodiments of any one of the above Formulae XIand XII, at least one of R¹⁰ and R¹¹ is C₂ alkyl. In certain embodimentsof any one of the above Formulae XI and XII, at least one of R¹⁰ and R¹¹is C₃ alkyl. In certain embodiments of any one of the above Formulae XIand XII, at least one of R¹⁰ and R¹¹ is C₄ alkyl.

In certain embodiments of any one of the above Formulae XI and XII, atleast one of R¹⁰ and R¹¹ is alkyl substituted with aryl, aryloxy,heteroaryl, heteroaryloxy, heterocyclic, or heterocyclooxy. In certainembodiments of any one of the above Formulae XI and XII, at least one ofR¹⁰ and R¹¹ is alkyl substituted with aryl, heteroaryl, or heterocyclic.In certain embodiments of any one of the above Formulae XI and XII, atleast one of R¹⁰ and R¹¹ is alkyl substituted with aryl. In certainembodiments of any one of the above Formulae XI and XII, at least one ofR¹⁰ and R¹¹ is alkyl substituted with heteroaryl. In certain embodimentsof any one of the above Formulae XI and XII, at least one of R¹⁰ and R¹¹is alkyl substituted with heterocyclic.

In some embodiments, a subject synthetic regulator of polypeptidefunction is a compound of Formula I:

wherein each of R¹ are independently selected from hydrogen, C₁₋₁₀alkyl, substituted C₁₋₁₀ alkyl, —NR¹⁰R¹¹, —NR¹²C(O)R¹³, C₂₋₁₀ alkenyl,substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substituted C₂₋₁₀ alkynyl,C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl, heteroaryl, heterocyclic,heterocyclooxy, heterocyclothio, heteroarylamino, heterocycloamino,C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl,substituted C₄₋₁₀ cycloalkenyl, cyano, halo, —OR¹⁰, —C(O)OR¹⁰, SR¹⁰,—S(O)R¹⁰, —S(O)₂R¹⁰;

x is an integer from 1 to 5;

R² is hydrogen or C₁₋₁₀ alkyl;

R³, R⁴, and R⁵ are independently selected from hydrogen and C₂₋₈ alkyl;

R¹⁰ and R¹¹ are independently selected from hydrogen and C₁₋₁₀ alkyl;

R¹² is hydrogen or C₁₋₁₀ alkyl;

R¹³ is selected from hydrogen, C₁₋₁₀ alkyl, C₁₋₈ alkenyl, C₆₋₁₀ aryl,and substituted C₁₋₁₀ alkyl,

or a pharmaceutically acceptable salt thereof.

In some embodiments, a subject synthetic regulator of polypeptidefunction is a compound of Formula II:

wherein each of R¹ are independently selected from hydrogen, C₁₋₁₀alkyl, —NR¹⁰R¹¹, —NR¹²C(O)R¹³, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, cyano,halo, —OR¹⁰, —C(O)OR¹⁰, —SR¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰;

x is an integer from 1 to 5;

R² is hydrogen or C₁₋₁₀ alkyl;

R³, R⁴, and R⁵ are independently selected from hydrogen and C₂₋₈ alkyl;

R¹⁰ and R¹¹ are independently selected from hydrogen and C₁₋₁₀ alkyl;

R¹² is hydrogen or C₁₋₁₀ alkyl;

R¹³ is selected from hydrogen, C₁₋₁₀ alkyl, C₁₋₈ alkenyl, C₆₋₁₀ aryl,and substituted C₁₋₁₀ alkyl,

or a pharmaceutically acceptable salt thereof.

In some embodiments, a subject synthetic regulator of polypeptidefunction is a compound of Formula III:

wherein each of R¹ are independently selected from hydrogen, C₁₋₁₀alkyl, —NR¹⁰R¹¹, and —NR¹²C(O)R¹³;

x is an integer from 1 to 5;

R² is hydrogen or C₁₋₁₀ alkyl;

R³, R⁴, and R⁵ are independently selected from hydrogen and C₂₋₈ alkyl;

R¹⁰ and R¹¹ are independently selected from hydrogen and C₁₋₁₀ alkyl;

R¹² is hydrogen or C₁₋₁₀ alkyl;

R¹³ is selected from hydrogen, C₁₋₁₀ alkyl, C₁₋₈ alkenyl, C₆₋₁₀ aryl,and substituted C₁₋₁₀ alkyl,

or a pharmaceutically acceptable salt thereof.

In some embodiments, a subject synthetic regulator of polypeptidefunction is a compound of Formula IV:

wherein each of R¹ are independently selected from hydrogen, C₁₋₁₀alkyl, —NR¹⁰R¹¹, and —NR¹²C(O)R¹³;

x is an integer from 1 to 5;

R² is hydrogen or C₁₋₁₀ alkyl;

R³, R⁴, and R⁵ are independently selected from hydrogen and C₂₋₈ alkyl;

R¹⁰ and R¹¹ are independently selected from hydrogen and C₁₋₁₀ alkyl;

R¹² is hydrogen or C₁₋₁₀ alkyl;

R¹³ is selected from hydrogen, C₁₋₁₀ alkyl, C₁₋₈ alkenyl, C₆₋₁₀ aryl,and substituted C₁₋₁₀ alkyl,

or a pharmaceutically acceptable salt thereof.

In some embodiments, a subject synthetic regulator of polypeptidefunction is a compound of Formula V:

wherein R¹ is selected from hydrogen, C₁₋₁₀ alkyl, —NR¹⁰R¹¹, and—NR¹²C(O)R¹³;

R² is hydrogen or C₁₋₁₀ alkyl;

R³, R⁴, and R⁵ are independently selected from hydrogen and C₂₋₈ alkyl;

R¹⁰ and R¹¹ are independently selected from hydrogen and C₁₋₁₀ alkyl;

R¹² is hydrogen or C₁₋₁₀ alkyl;

R¹³ is selected from hydrogen, C₁₋₁₀ alkyl, C₁₋₈ alkenyl, C₆₋₁₀ aryl,and substituted C₁₋₁₀ alkyl,

or a pharmaceutically acceptable salt thereof.

In some embodiments, a subject synthetic regulator of polypeptidefunction is a compound of Formula VI:

wherein R¹ is selected from hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀alkyl, —NR¹⁰R¹¹, —NR¹²C(O)R¹³, C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl,C₂₋₁₀ alkynyl, substituted C₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆₋₂₀aryl, heteroaryl, heterocyclic, heterocyclooxy, heterocyclothio,heteroarylamino, heterocycloamino, C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀cycloalkyl, C₄₋₁₀ cycloalkenyl, substituted C₄₋₁₀ cycloalkenyl, cyano,halo, —OR¹⁰, —C(O)OR¹⁰, —SR¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰;

R² is hydrogen or C₁₋₁₀ alkyl;

R³, R⁴, and R⁵ are independently selected from hydrogen and C₂₋₈ alkyl;

R¹⁰ and R¹¹ are independently selected from hydrogen and C₁₋₁₀ alkyl;

R¹² is hydrogen or C₁₋₁₀ alkyl;

R¹³ is selected from hydrogen, C₁₋₁₀ alkyl, C₁₋₈ alkenyl, C₆₋₁₀ aryl,and substituted C₁₋₁₀ alkyl,

or a pharmaceutically acceptable salt thereof. In some embodiments, acompound of Formula VI has no carbonyl group.

In some embodiments, a subject synthetic regulator of polypeptidefunction is a compound of Formula VII:

wherein R¹ is selected from hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀alkyl, —NR¹⁰R¹¹, —NR¹²C(O)R¹³, C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl,C₂₋₁₀ alkynyl, substituted C₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆₋₂₀aryl, heteroaryl, heterocyclic, heterocyclooxy, heterocyclothio,heteroarylamino, heterocycloamino, C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀cycloalkyl, C₄₋₁₀ cycloalkenyl, substituted C₄₋₁₀ cycloalkenyl, cyano,halo, —OR¹⁰, —C(O)OR¹⁰, —SR¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰;

x is an integer from 1 to 4;

R² is hydrogen or C₁₋₁₀ alkyl;

R³, R⁴, and R⁵ are independently selected from hydrogen and C₂₋₈ alkyl;

each of R⁶ and R⁷ are independently selected from hydrogen. C₁₋₁₀ alkyl,substituted C₁₋₁₀ alkyl, —NR¹⁰R¹¹; —NR¹²C(O)R¹³, C₂₋₁₀ alkenyl,substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substituted C₂₋₁₀ alkynyl,C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl, heteroaryl, heterocyclic,heterocyclooxy, heterocyclothio, heteroarylamino, heterocycloamino,C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl,substituted C₄₋₁₀ cycloalkenyl, cyano, halo, —OR¹⁰, —C(O)OR¹⁰, —SR¹⁰,—S(O)R¹⁰, —S(O)₂R¹⁰;

R¹⁰ and R¹¹ are independently selected from hydrogen and C₁₋₁₀ alkyl;

R¹² is hydrogen or C₁₋₁₀ alkyl;

R¹³ is selected from hydrogen, C₁₋₁₀ alkyl, C₁₋₈ alkenyl, C₆₋₁₀ aryl,and substituted C₁₋₁₀ alkyl,

or a pharmaceutically acceptable salt thereof.

In some embodiments, a subject synthetic regulator of polypeptidefunction is a compound of Formula VIII:

wherein R¹ is selected from hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀alkyl, —NR¹⁰R¹¹, —NR¹²C(O) R¹³, C₂₋₁₀ alkenyl, substituted C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, substituted C₂₋₁₀ alkynyl, C₆₋₂₀ aryl,substituted C₆₋₂₀ aryl, heteroaryl, heterocyclic, heterocyclooxy,heterocyclothio, heteroarylamino, heterocycloamino, C₄₋₁₀ cycloalkyl,substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl, substituted C₄₋₁₀cycloalkenyl, cyano, halo, —OR¹⁰, —C(O)OR¹⁰, —SR¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰;

x is an integer from 1 to 4;

R² is hydrogen or C₁₋₁₀ alkyl;

R³ and R⁴ are independently selected from hydrogen and C₂₋₈ alkyl;

each of R⁶ and R⁷ are independently selected from hydrogen, C₁₋₁₀ alkyl,substituted C₁₋₁₀ alkyl, —NR¹⁰R¹¹, —NR¹²C(O)R¹³, C₂₋₁₀ alkenyl,substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substituted C₂₋₁₀ alkynyl,C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl, heteroaryl, heterocyclic,heterocyclooxy, heterocyclothio, heteroarylamino, heterocycloamino,C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl,substituted C₄₋₁₀ cycloalkenyl, cyano, halo, —OR¹⁰, —C(O)OR¹⁰, —SR¹⁰,—S(O)R¹⁰, —S(O)₂R¹⁰;

R¹⁰ and R¹¹ are independently selected from hydrogen and C₁₋₁₀ alkyl;

R¹² is hydrogen or C₁₋₁₀ alkyl;

R¹³ is selected from hydrogen, C₁₋₁₀ alkyl, C₁₋₈ alkenyl, C₆₋₁₀ aryl,and substituted C₁₋₁₀ alkyl,

or a pharmaceutically acceptable salt thereof. In some embodiments, thenitrogen is not permanently charged.

In some embodiments, a subject synthetic regulator of polypeptidefunction is a compound of Formula IX:

wherein

R¹ is selected from hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀ alkyl,—NR¹⁰R¹¹, —NR¹²C(O)R¹³, C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, substituted C₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl,heteroaryl, heterocyclic, heterocyclooxy, heterocyclothio,heteroarylamino, heterocycloamino, C₄₋₁₀ cycloalkyl, cycloalkyl,substituted C₄₋₁₀ cycloalkenyl, substituted C₄₋₁₀ cycloalkenyl, cyano,halo, —OR¹⁰, —C(O)OR¹⁰, —SR¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰;

x is an integer from 1 to 4;

y is an integer from 1 to 4;

R² is hydrogen or C₁₋₁₀ alkyl;

R³, R⁴, and R⁶ are independently selected from hydrogen and C₂₋₈ alkyl;

each of R⁶ and R⁷ are independently selected from hydrogen. C₁₋₁₀ alkyl,substituted C₁₋₁₀ alkyl, NR¹⁰R¹¹, —NR¹²C(O)R¹³, C(O)R¹³, C₂₋₁₀ alkenyl,substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substituted C₂₋₁₀ alkynyl,C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl, heteroaryl, heterocyclic,heterocyclooxy, heterocyclothio, heteroarylamino, heterocycloamino,C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl,substituted C₄₋₁₀ cycloalkenyl, cyano, halo, —OR¹⁰, —C(O)OR¹⁰, —SR¹⁰,—S(O)R¹⁰, —S(O)₂R¹⁰;

R¹⁰ and R¹¹ are independently selected from hydrogen and C₁₋₁₀ alkyl;

R¹² is hydrogen or C₁₋₁₀ alkyl;

R¹³ is selected from hydrogen, C₁₋₁₀ alkyl, C₁₋₈ alkenyl, C₆₋₁₀ aryl,and substituted C₁₋₁₀ alkyl,

or a pharmaceutically acceptable salt thereof.

In some embodiments, a subject synthetic regulator of polypeptidefunction is a compound of Formula X:

wherein

R¹ is selected from hydrogen, C₁₋₁₀ alkyl, substituted C₁₋₁₀ alkyl,—NR¹⁰R¹¹, —NR¹²C(O)R¹³, C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, substituted C₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl,heteroaryl, heterocyclic, heterocyclooxy, heterocyclothio,heteroarylamino, heterocycloamino, C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀cycloalkyl, C₄₋₁₀ cycloalkenyl, substituted C₄₋₁₀ cycloalkenyl, cyano,halo, —OR¹⁰, —C(O)OR¹⁰, —SR¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰;

x is an integer from 1 to 4;

y is an integer from 1 to 4;

R² is hydrogen or C₁₋₁₀ alkyl;

R³ and R⁴ are independently selected from hydrogen and C₂₋₈ alkyl;

each of R⁶ and R⁷ are independently selected from hydrogen. C₁₋₁₀ alkyl,substituted C₁₋₁₀ alkyl, —NR¹⁰R¹¹, —NR¹²C(O)R¹³, C₂₋₁₀ alkenyl,substituted C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substituted C₂₋₁₀ alkynyl,C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl, heteroaryl, heterocyclic,heterocyclooxy, heterocyclothio, heteroarylamino, heterocycloamino,C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀ cycloalkyl, C₄₋₁₀ cycloalkenyl,substituted C₄₋₁₀ cycloalkenyl, cyano, halo, —OR¹⁰, —C(O)OR¹⁰, —SR¹⁰,—S(O)R¹⁰, —S(O)₂R¹⁰;

R¹⁰ and R¹¹ are independently selected from hydrogen and C₁₋₁₀ alkyl;

R¹² is hydrogen or C₁₋₁₀ alkyl;

R¹³ is selected from hydrogen, C₁₋₁₀ alkyl, C₁₋₈ alkenyl, C₆₋₁₀ aryl,and substituted C₁₋₁₀ alkyl,

or a pharmaceutically acceptable salt thereof.

In certain embodiments of any one of the above Formulas I-X, R² ishydrogen. In certain embodiments of any one of the above Formulas I-X,R² is C₁₋₁₀ alkyl. In certain embodiments of any one of the aboveFormulas I-X, R² is C₁₋₅ alkyl. In certain embodiments of any one of theabove Formulas I-X, R² is hydrogen or C₁₋₅ alkyl.

In certain embodiments of any one of the above Formulas I-X, R¹ ishydrogen.

In certain embodiments of any one of the above Formulas I-X, R¹ is C₁₋₈alkyl, e.g., C₁₋₆ alkyl, C₁₋₅ alkyl or C₁₋₄ alkyl. In some embodimentsof any one of the above Formulas I-X, R¹ is C₁₋₄ alkyl.

In certain embodiments of any one of the above Formulas I-X, R¹ is—NR¹²C(O)R¹³.

In certain embodiments of any one of the above Formulas I-X, R¹² ishydrogen. In certain embodiments of any one of the above Formulas I-X,R¹² is C₁₋₁₀ alkyl. In certain embodiments of any one of the aboveFormulas I-X, R¹² is C₁₋₅ alkyl. In certain embodiments of any one ofthe above Formulas I-X, R¹² is hydrogen or C₁₋₅ alkyl.

In certain embodiments of any one of the above Formulas I-X, R¹³ ishydrogen or C₁₋₁₀ alkyl. In certain embodiments of any one of the aboveFormulas I-X, R¹³ is C₁₋₁₀ alkyl. In certain embodiments of any one ofthe above Formulas I-X, R¹³ is C₁₋₅ alkyl. In certain embodiments of anyone of the above Formulas I-X, R¹³ is hydrogen or C₁₋₅ alkyl.

In certain embodiments of any one of the above Formulas I-X, R¹³ isalkenyl or substituted alkenyl. In certain embodiments of any one of theabove Formulas I-X, R¹³ is alkenyl. In certain embodiments of any one ofthe above Formulas I-X, R¹³ is C₁₋₅ alkenyl. In certain embodiments ofany one of the above Formulas I-X, R¹³ is hydrogen or C₁₋₅ alkenyl.

In certain embodiments of any one of the above Formulas I-X, R¹³ is C₆aryl or substituted C₆ aryl.

In certain embodiments of any one of the above Formulas I-X, R¹³ isalkyl substituted with SO₃H, —SO₃ ⁻, —NR_(a)R_(b), —N⁺R_(a)R_(b)R_(c),wherein R_(a), R_(b), and R_(c) may be the same or different and arechosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic. In certainembodiments of any one of the above Formulas I-X, R¹³ is alkylsubstituted with SO₃H or —SO₃ ⁻. In certain embodiments of any one ofthe above Formulas I-X, R¹³ is alkyl substituted with —NR_(a)R_(b) or—N⁺R_(a)R_(b)R_(c), In certain embodiments of any one of the aboveFormulas I-X, R¹³ is alkyl substituted with —NR_(a)R_(b) or—N⁺R_(a)R_(b)R_(c), and wherein R_(a), R_(b), and R_(c) may be the sameor different and are chosen from hydrogen and optionally substitutedalkyl. In certain embodiments of any one of the above Formulas I-X, R¹³is alkyl substituted with —NR_(a)R_(b) or —N⁺R_(a)R_(b)R_(c), andwherein R_(a), R_(b), and R_(c) are alkyl.

In some embodiments, a subject synthetic regulator is a non-permanentlycharged compound. In some embodiments, a subject synthetic regulatorcomprises a substituted azobenzene group. In some embodiments, a subjectsynthetic regulator is a cis blocker, e.g., blocks a receptor (such asan ion channel) when in the cis isomeric form. In other embodiments, asubject synthetic regulator is a trans-blocker blocker, e.g., blocks areceptor (such as an ion channel) when in the trans isomeric form.

In some embodiments, a subject synthetic regulator acts on more than onepolypeptide. For example, QAQ blocks voltage-gated potassium channels(K_(ν)), voltage-gated sodium channels (Na_(ν)), and voltage-gatedcalcium channels (Ca_(ν)) channels. In other embodiments, a subjectsynthetic regulator exhibits selectivity, e.g., in some embodiments, asubject synthetic regular selectively blocks a voltage-gated potassiumchannel, but does not substantially block a voltage-gated sodium channelor a voltage-gated calcium channel.

In some embodiments, a subject synthetic regulator comprises ared-shifted photoisomerizable group, e.g., the photoisomerizable groupof a synthetic regulator is in a first isomeric form when exposed to afirst wavelength of light, and is in a second isomeric form when exposedto a second wavelength of light, where the second wavelength is shiftedtoward the red end of the spectrum compared to the first wavelength oflight. As an example, DAAQ is in a first isomeric form at 472 nm and ina second isomeric form at 550 nm.

In some embodiments, a subject synthetic regulator is membrane permeant,e.g., will cross a eukaryotic cell membrane without the need for anyadditional physical, electrical, or chemical stimulus to be applied tothe cell.

In some embodiments, a subject synthetic regulator is membraneimpermeant; for example, in some embodiments, a subject syntheticregulator enters a eukaryotic cell only upon application of anadditional physical, electrical, or chemical stimulus to the cell. Forexample, in some embodiments, a subject synthetic regulator enters aeukaryotic cell (e.g., a neuron) only upon application of a physical,electrical, or chemical stimulus that activates a nonselective ionchannel. Nonselective ion channels include, e.g., ligand-gatednonselective cation channels. Nonselective cation channels include,e.g., TRPV₁, P2X₇R, and the like. P2X₇R (or P2X purinoceptor 7) isdescribed in, e.g., Chessell et al. (2005) Pain 114:386; and Rassendrenet al. (1997) J. Biol. Chem. 272:5482. P2X₇R can be activated byadenosine triphosphate (ATP), or an ATP analog. An example of amembrane-impermeant synthetic regulator is QAQ.

TRPV₁ (transient receptor potential cation channel, subfamily V, member1; also known as vanilloid receptor type 1), is a ligand-gatednonselective cation channel that is activated by a variety of endogenousand exogenous physical and chemical stimuli, including, e.g., heat over43° C., low pH, the endocannabinoid anandamide, N-arachidonoyl-dopamine,and capsaicin. For TRPV₁, see, e.g., Cui et al. (2006) J. Neurosci.26:9385.

TRPV₁ agonists include, e.g., capsaicin; a capsaicinoid (wherecapsaicinoids include, e.g., capsiate (4-hydroxy-3-methoxybenzyl(E)-8-methyl-6-nonenoate); dihydrocapsiate (4-hydroxy-3-methoxybenzyl8-methylnonanoate); nordihydrocapsiate (4-hydroxy-3-methoxybenzyl7-methyl-octanoate); capsiate derivatives such as vanillyl decanoate,vanillyl nonanoate, vanillyl octanoate and the like; fatty acid estersof vanillyl alcohol; and various straight chain or branched chain fattyacids which have a fatty acid chain length similar to that ofnordihydrocapsiate); resiniferatoxin; olvanil; tinyatoxin; a compound asdescribed in U.S. Patent Publication No. 2006/0240097; a compound asdescribed in U.S. Patent Publication No. 2009/0203774; a pentadienamidederivative as described in U.S. Patent Publication No. 2009/0203667; acompound as described in U.S. Patent Publication No. 2009/0170942; andthe like.

Exemplary synthetic regulators include compounds of the followingstructures:

As described above, the present disclosure provides a syntheticregulator of protein function. A subject synthetic regulator of proteinfunction is useful for regulating protein function by use of light. Asubject synthetic regulator can be provided in any number ofconfigurations, including linear and branched, which can be affected bylight.

For example, the configuration of BzAQ can change with application ofcertain wavelengths of light.

Other characteristics of BzAQ include being a trans-blocker, an externalblocker, and selective for K_(ν) channels.

In another example, the configuration of BEAAQ can change withapplication of certain wavelengths of light.

Other characteristics of BEAAQ include being a cis-blocker and beingable to block K_(ν) channels.

In another example, the configuration of DAAQ can change withapplication of certain wavelengths of light.

Other characteristics of DAAQ include being a trans-blocker, an externalblocker, a red-shifted compound, and being able to block K_(ν) channels.

In another example, the configuration of QAQ can change with applicationof certain wavelengths of light.

Other characteristics of QAQ include being a trans-blocker, an internalblocker, and being able to block K_(ν), Na_(ν), and Ca_(ν) channels.Compositions

The embodiments further provide compositions comprising a subjectsynthetic regulator. Compositions comprising a subject syntheticregulator can include one or more of: a salt, e.g., NaCl, MgCl, KCl,MgSO₄, etc.; a buffering agent, e.g., a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-morpholino)ethanesulfonic acid (MES),2-(N-morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; asolubilizing agent; a detergent, e.g., a non-ionic detergent such asTween-20, Nonidet-P40, etc.: a protease inhibitor; and the like.

Pharmaceutical Compositions

The embodiments provide pharmaceutical compositions comprising a subjectsynthetic regulator. In some embodiments, the pharmaceutical compositionis suitable for administering to an individual in need thereof.

A pharmaceutical composition comprising a subject synthetic regulatormay be administered to a patient alone, or in combination with othersupplementary active agents. The pharmaceutical compositions may bemanufactured using any of a variety of processes, including, withoutlimitation, conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping, and lyophilizing.The pharmaceutical composition can take any of a variety of formsincluding, without limitation, a sterile solution, suspension, emulsion,lyophilisate, tablet, pill, pellet, capsule, powder, syrup, elixir orany other dosage form suitable for administration.

A pharmaceutical composition comprising a subject synthetic regulatorcan optionally include a pharmaceutically acceptable carrier(s) thatfacilitate processing of an active ingredient into pharmaceuticallyacceptable compositions. As used herein, the term “pharmacologicallyacceptable carrier” refers to any carrier that has substantially nolong-term or permanent detrimental effect when administered andencompasses terms such as “pharmacologically acceptable vehicle,stabilizer, diluent, auxiliary or excipient.” Such a carrier generallyis mixed with an active compound, or permitted to dilute or enclose theactive compound and can be a solid, semi-solid, or liquid agent. It isunderstood that the active ingredients can be soluble or can bedelivered as a suspension in the desired carrier or diluent. Any of avariety of pharmaceutically acceptable carriers can be used including,without limitation, aqueous media such as, e.g., distilled, deionizedwater, saline; solvents; dispersion media; coatings; antibacterial andantifungal agents; isotonic and absorption delaying agents; or any otherinactive ingredient. Selection of a pharmacologically acceptable carriercan depend on the mode of administration. Except insofar as anypharmacologically acceptable carrier is incompatible with the activeingredient, its use in pharmaceutically acceptable compositions iscontemplated. Non-limiting examples of specific uses of suchpharmaceutical carriers can be found in “Pharmaceutical Dosage Forms andDrug Delivery Systems” (Howard C. Ansel et al., eds., LippincottWilliams & Wilkins Publishers, 7^(th) ed. 1999); “Remington: The Scienceand Practice of Pharmacy” (Alfonso R. Gennaro ed., Lippincott, Williams& Wilkins, 20^(th) 2000); “Goodman & Gilman's The Pharmacological Basisof Therapeutics” Joel G. Hardman et al., eds., McGraw-Hill Professional,10.sup.th ed. 2001); and “Handbook of Pharmaceutical Excipients”(Raymond C. Rowe et al., APhA Publications, 4^(th) edition 2003).

A subject pharmaceutical composition can optionally include, withoutlimitation, other pharmaceutically acceptable components, including,without limitation, buffers, preservatives, tonicity adjusters, salts,antioxidants, physiological substances, pharmacological substances,bulking agents, emulsifying agents, wetting agents, sweetening orflavoring agents, and the like. Various buffers and means for adjustingpH can be used to prepare a pharmaceutical composition disclosed in thepresent specification, provided that the resulting preparation ispharmaceutically acceptable. Such buffers include, without limitation,acetate buffers, citrate buffers, phosphate buffers, neutral bufferedsaline, phosphate buffered saline and borate buffers. It is understoodthat acids or bases can be used to adjust the pH of a composition asneeded. Pharmaceutically acceptable antioxidants include, withoutlimitation, sodium metabisulfite, sodium thiosulfate, acetylcysteine,butylated hydroxyanisole and butylated hydroxytoluene. Usefulpreservatives include, without limitation, benzalkonium chloride,chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuricnitrate and a stabilized oxy chloro composition, for example, PURITE™.Tonicity adjustors suitable for inclusion in a subject pharmaceuticalcomposition include, without limitation, salts such as, e.g., sodiumchloride, potassium chloride, mannitol or glycerin and otherpharmaceutically acceptable tonicity adjustor. It is understood thatthese and other substances known in the art of pharmacology can beincluded in a subject pharmaceutical composition.

Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

A subject synthetic regulator can be formulated with one or morepharmaceutically acceptable excipients. A wide variety ofpharmaceutically acceptable excipients are known in the art and need notbe discussed in detail herein. Pharmaceutically acceptable excipientshave been amply described in a variety of publications, including, forexample, A. Gennaro (2000) “Remington: The Science and Practice ofPharmacy,” 20th edition, Lippincott, Williams, & Wilkins; PharmaceuticalDosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds.,7^(th) ed., Lippincott, Williams, & Wilkins; and Handbook ofPharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3^(rd) ed.Amer. Pharmaceutical Assoc.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

In the subject methods (described below), a subject synthetic regulatormay be administered to the host using any convenient means capable ofresulting in the desired reduction in disease condition or symptom.Thus, a subject synthetic regulator can be incorporated into a varietyof formulations for therapeutic administration. More particularly, asubject synthetic regulator can be formulated into pharmaceuticalcompositions by combination with appropriate pharmaceutically acceptablecarriers or diluents, and may be formulated into preparations in solid,semi-solid, liquid or gaseous forms, such as tablets, capsules, powders,granules, ointments, solutions, suppositories, injections, inhalants andaerosols.

A subject synthetic regulator can be used alone or in combination withappropriate additives to make tablets, powders, granules or capsules,for example, with conventional additives, such as lactose, mannitol,corn starch or potato starch; with binders, such as crystallinecellulose, cellulose derivatives, acacia, corn starch or gelatins; withdisintegrators, such as corn starch, potato starch or sodiumcarboxymethylcellulose; with lubricants, such as talc or magnesiumstearate; and if desired, with diluents, buffering agents, moisteningagents, preservatives and flavoring agents. Such preparations can beused for oral administration.

A subject synthetic regulator can be formulated into preparations forinjection by dissolving, suspending or emulsifying them in an aqueous ornonaqueous solvent, such as vegetable or other similar oils, syntheticaliphatic acid glycerides, esters of higher aliphatic acids or propyleneglycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives. Formulations suitable for injection canbe administered by an intravitreal, intraocular, intramuscular,subcutaneous, sublingual, or other route of administration, e.g.,injection into the gum tissue or other oral tissue. Such formulationsare also suitable for topical administration.

A subject synthetic regulator can be utilized in aerosol formulation tobe administered via inhalation. A subject synthetic regulator can beformulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, a subject synthetic regulator can be made intosuppositories by mixing with a variety of bases such as emulsifyingbases or water-soluble bases. A subject synthetic regulator can beadministered rectally via a suppository. The suppository can includevehicles such as cocoa butter, carbowaxes and polyethylene glycols,which melt at body temperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise a subject synthetic regulator in acomposition as a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of a subjectsynthetic regulator calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for a subject syntheticregulator depend on the particular compound employed and the effect tobe achieved, and the pharmacodynamics associated with each compound inthe host.

A subject synthetic regulator can be administered as injectables.Injectable compositions are prepared as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid vehiclesprior to injection may also be prepared. The preparation may also beemulsified or the active ingredient encapsulated in liposome vehicles.

In some embodiments, a subject synthetic regulator is delivered by acontinuous delivery system. The term “continuous delivery system” isused interchangeably herein with “controlled delivery system” andencompasses continuous (e.g., controlled) delivery devices (e.g., pumps)in combination with catheters, injection devices, and the like, a widevariety of which are known in the art.

Light-Regulated Polypeptides

The present disclosure further provides a light-regulated polypeptide,where a subject light-regulated polypeptide comprises a polypeptide anda subject synthetic regulator of receptor function in non-covalentassociation with the polypeptide. The synthetic regulator of polypeptidefunction can be non-covalently associated with the polypeptide at ornear a ligand binding site of the polypeptide. In some embodiments, asubject light-regulated polypeptide is isolated, e.g., free of otherpolypeptides or other macromolecules. In other embodiments, a subjectlight-regulated polypeptide is membrane-associated and is present invitro. In other embodiments, a subject light-regulated polypeptide ispresent in a living cell in vitro or in vivo. In other embodiments, asubject light-regulated polypeptide is present in a membrane of a livingcell in vitro or in vivo. In other embodiments, a subjectlight-regulated polypeptide is present in a living cell in a tissue invitro or in vivo. In other embodiments, a subject light-regulatedpolypeptide is present in a living cell in a multicellular organism.Polypeptides with which a subject synthetic regulator can benon-covalently associated include, e.g., receptors, ion channels,enzymes, and the like.

A change in the wavelength and/or intensity of light (Δλ) to which thelight-regulated polypeptide is exposed results in a change in ligandbinding to a ligand-binding site of the light-regulated polypeptide,e.g., results in a change in binding of the ligand portion of thesynthetic polypeptide to the ligand-binding site of the light-regulatedpolypeptide. A “change in the wavelength of light to which thelight-regulated polypeptide is exposed” includes: 1) a change from λ₁ toλ₂; 2) a change from λ₂ to λ₁; 3) a change from λ₁ to darkness (nolight); and 4) a change from darkness to λ₁. Repetitive changing from λ₁to λ₂, then from λ₂ to λ₁, and back, e.g., switching from a firstwavelength to a second wavelength, and back again repeatedly, is alsocontemplated. Repetitive changing from light to darkness, from darknessto light, etc., is also contemplated.

In some embodiments, the change in wavelength (from λ₁ to λ₂; from lightto darkness; or from darkness to light) results in a change in bindingof the ligand to a ligand-binding site. As used herein, a “change inbinding of a ligand to a ligand-binding site” includes increased bindingand decreased binding. As used herein, “increased binding” includes oneor more of: an increased probability of binding of the ligand to theligand-binding site; an increased binding affinity of the ligand for theligand-binding site; an increased local concentration of the ligand atthe ligand-binding site; and an increased occupancy of the ligand in theligand-binding site. As used herein, “decreased binding” includes one ormore of: a decreased probability of binding of the ligand to theligand-binding site; a decreased binding affinity of the ligand for theligand-binding site; a decreased local concentration of the ligand atthe ligand-binding site; and a decreased occupancy of the ligand in theligand-binding site. As used herein, the term “change in wavelength” towhich a synthetic regulator is exposed, or to which a receptor/syntheticlight regulator complex is exposed, refers to a change in wavelengthfrom λ₁ to λ₂; a change from light to darkness; or a change fromdarkness to light. An increase in binding includes an increase of fromabout 10% to about 50%, from about 50% to about 2-fold, from about2-fold to about 5-fold, from about 5-fold to about 10-fold, from about10-fold to about 50-fold, from about 50-fold to about 10²-fold, fromabout 10²-fold to about 10⁴-fold, from about 10⁴-fold to about 10⁶-fold,from about 10⁶-fold to about 10⁸-fold, or a greater than 10⁸-foldincrease in binding. A decrease in binding includes a decrease of fromabout 5% to about 10% to about 20% to about 30%, from about 30% to about40%, from about 40% to about 50%, from about 50% to about 60%, fromabout 60% to about 70%, from about 70% to about 80%, from about 80% toabout 90%, or from about 90% to 100% decrease in binding.

For example, in some embodiments, the ligand has a first probability ofbinding to the ligand site at a first wavelength of light; the ligandhas a second probability of binding to the ligand binding site at asecond wavelength of light; and the second probability is lower than thefirst probability. In other embodiments, the ligand has a firstprobability of binding to the ligand site at a first wavelength oflight; the ligand has a second probability of binding to the ligandbinding site at a second wavelength of light; and the second probabilityis higher than the first probability. In other embodiments, ligand has afirst probability of binding to the ligand site when exposed to light;the ligand has a second probability of binding to the ligand bindingsite in the absence of light (e.g., in darkness); and the secondprobability is lower than the first probability. In other embodiments,the ligand has a first probability of binding to the ligand site whenexposed to light; the ligand has a second probability of binding to theligand binding site in the absence of light and the second probabilityis higher than the first probability.

The local concentration of the ligand portion of the synthetic regulatorat the ligand binding site in a subject light-regulated polypeptide ishigh. For example, the local concentration of the ligand portion of thesynthetic regulator at the ligand binding site in a subjectlight-regulated polypeptide ranges from about 500 nM to about 50 mM,e.g., from about 500 nM to about 750 nM, from about 750 nM to about 1mM, from about 1 mM to about 5 mM, from about 5 mM to about 10 mM, fromabout 10 mM to about 20 mM, from about 20 mM to about 30 mM, or fromabout 30 mM to about 50 mM.

Change in Wavelength Resulting in Binding of the Ligand to theLigand-Binding Site or Higher Affinity Ligand Binding to Ligand-BindingSite

In some embodiments, a change in the wavelength of light to which thelight-regulated polypeptide is exposed results in an increase in bindingaffinity of the ligand portion of a subject synthetic regulator for aligand-binding site of the polypeptide portion of the light-regulatedpolypeptide. For example, in some embodiments, a change in wavelength oflight to which the light-regulated polypeptide is exposed results in anat least about 10%, at least about 20%, at least about 30%, at leastabout 50%, at least about 75%, at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 25-fold, at least about50-fold, at least about 100-fold, at least about 250-fold, at leastabout 500-fold, at least about 10³-fold, at least about 5×10³-fold, atleast about 10⁴-fold, at least about 5×10⁴-fold, or greater, increase inbinding affinity.

Where the ligand is an agonist, the change in wavelength will in someembodiments result in activation of the light-regulated polypeptide.Where the ligand is an agonist, the change in wavelength will in someembodiments result in desensitization of the light-regulatedpolypeptide. Conversely, where the ligand is an antagonist, the changein wavelength results in a block of activation of the light-regulatedpolypeptide, e.g., block of the ability to activate the light-regulatedpolypeptide with free agonist. Where the ligand is a blocker (e.g., apore blocker of an ion channel, or an interaction domain that binds toother biological macromolecules such as polypeptides or nucleic acids),the change in wavelength results in block of polypeptide activity.

Expressed another way, where the ligand is an agonist, and where achange in the wavelength of light to which the light-regulatedpolypeptide is exposed results in a higher binding affinity of theligand moiety of the synthetic regulator to the ligand-binding site ofthe light-regulated polypeptide, the change in wavelength results intransition from an inactive state to an active state, or to adesensitized state. Where the ligand is an antagonist, the change inwavelength results in transition from a responsive state to anunresponsive state. Where the ligand is a blocker, the change inwavelength results in transition from an active state to an inactivestate.

Change in Wavelength Resulting in Removal of Ligand from Ligand-BindingSite, or Reduced Binding Affinity

In some embodiments, a change in the wavelength of light to which thelight-regulated polypeptide is exposed results in removal of the ligandportion of a subject synthetic regulator from a ligand-binding site ofthe light-regulated polypeptide, e.g., the ligand is not bound to theligand-binding site. In some embodiments, a change in the wavelength oflight to which the light-regulated polypeptide is exposed results inreduced binding affinity of the ligand portion of a subject syntheticregulator for a ligand-binding site of the light-regulated polypeptide,e.g., the ligand has reduced binding affinity for the ligand-bindingsite. For example, in some embodiments, a change in the wavelength oflight to which the light-regulated polypeptide is exposed results in areduction of binding affinity of at least about 10%, at least about 20%,at least about 25%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, or more.

Where the ligand is an agonist, the change in wavelength will in someembodiments result in deactivation of the light-regulated polypeptide.Where the ligand is an agonist, the change in wavelength will in someembodiments result in recovery from desensitization of thelight-regulated polypeptide. Conversely, where the ligand is anantagonist, the change in wavelength results in activation of thelight-regulated polypeptide, or results in removal of a blocker from thelight-regulated polypeptide. Where the ligand is a blocker (e.g., a poreblocker of an ion channel, or an interaction domain that binds to otherbiological macromolecules such as polypeptides or nucleic acids), thechange in wavelength results in relief of a block in polypeptideactivity and permits the receptor to function normally.

Expressed another way, where the ligand is an agonist, and where achange in the wavelength of light to which the light-regulatedpolypeptide is exposed results in removal (or non-binding) of the ligandmoiety of the synthetic regulator from the ligand-binding site of thelight-regulated polypeptide, the change in wavelength results intransition from an active state to an inactive state, or from adesensitized state to a responsive state. Where the ligand is anantagonist, the change in wavelength results in transition from anunresponsive state to a responsive state. Where the ligand is a blocker,the change in wavelength results in transition from an inactive state toan active state.

Ion Channels

In some embodiments, the polypeptide portion of a subjectlight-regulated polypeptide is an ion channel. A subject light-regulatedion channel comprises an ion channel and a subject synthetic regulatorof receptor function in non-covalent association with the ion channel.The synthetic regulator of polypeptide function is non-covalentlyassociated with the ion channel at or near a ligand binding site of thereceptor on the ion channel. In some embodiments, the syntheticregulator can provide occlusion to the ion channel. Ion channels withwhich a subject synthetic regulator of polypeptide function can benon-covalently associated include, e.g., sodium channels, potassiumchannels, calcium channels, and chloride channels. The ion channel canbe voltage regulated, cAMP regulated, or ligand gated.

Sodium Channels

A variety of different isoforms of mammalian voltage dependent sodiumchannels have been identified, and are summarized below in Table 1.These channels can be classified into three main groups (for review seeGoldin, Annals N.Y. Academy of Sciences 868:38-50, 1999).

TABLE 1 Sodium Channel Sub-type Summary Channel Name & Sub-type/ TissueAccession Gene Symbol Alternate Names Distribution Number SCN1A (Nav1.1)Rat I (rat) CNS/PNS X03638 HBSCI (human) CNS X65362 GPB1 (guinea pig)CNS AF003372 SCN2A (Nav1.2) Rat (rat) CNS X03639 HBSCH (human) CNSX65361 HBA (human) CNS M94055 Nav1.2A Rat IIA CNS X61149 SCN3A(Nav1.3)Rat III (rat) CNS Y00766 SCN4A (Nav1.4) SkM1, μ1 (rat) Skeletal muscleM26643 SkM1, (human) Skeletal muscle N81758 SCN5A (Nav1.5) SkM2 (rat)Skeletal muscle/ M27902 RH1 (rat) Heart H1 (human) heart M77235 SCN8A(Nav1.6) NaCh6 (rat) CNS/PNS L39018 PN4a (rat) CNS/PNS AF049239A Scn8a(mouse) CNS U26707 ScnSa (human) CNS AF050736 CerIII (guinea pig) CNSAF003373 SCN9A (Nav1.7) PN1 (rat) PNS U79568 HNE-Na (human) ThyroidX82835 Nas (rabbit) Schwann cells U35238 SCN9A (Nav1.7) SNS (rat) PNSX92184 PN3 (rat) PNS U53833 SNS (mouse) PNS Y09108 SCN6A Nav2.1 Na2.1(human) Heart, uterus, muscle M91556 SCN7A Nav2.2 Na-G (rat) AstrocytesM96578 SCL11 (rat) PNS Y09165 nav2.3 Na2.3 (mouse) Heart, uterus, muscleL36179 Nav3.1 SCN1b Nβ1.1 β1 (rat) CNS M91808 β1 (human) CNS L10338SCH2b Nβ2.1 β2 (rat) CNS U37026 β2 (human) CNS AF007783Potassium Channels

Voltage-dependent potassium channels repolarize nerve and muscle cellsafter action potential depolarization. They also play importantregulatory roles in neural, muscular, secretory, and excretory systems.

A summary of the numerous potassium sub-types is presented in Table 2below.

TABLE 2 Potassium Channel Sub-type Summary Channel Sub-type/ AccessionName Alternate Names Number Reference ATP- regulated rKir.1 U12541 USPat. No. 5,356,775 (ROMK1) (rat) hKir1.1 US Pat. No. 5,882,873 (ROMK1)(human) Kir1.1 U73191 Kir1.3 U73193 II. Bcell US Pat. No. 5,744,594 III.hβIR US Pat. No. 5,917,027 IV. IIuK_(ATP)-1 EP0 768 379A1 Constitu-tively active Kir2.1 (IRK1) U12507 US Pat. No. 5,492,825 US Pat. No.5,670,335 Kir2.2 X78461 Kir2.3 X78461 G-protein regulated Kir3.1 (GIK1,KGA) U0171 US Pat. No. 5,728,535 Kir3.2 U11859 US Pat. No. 5,734,021Kir3.3 U11869 US Pat. No. 5,744,324 Kir3.4 (CIR) X83584 US Pat. No.5,747,278 Kir4.1(BIR10) X83585 Kir5.1(BIR9) X83581 Kir6.1 D42145 Kir6.2D5081 Kir7.1 EP0 922 763A1 Voltage regulated KCNA1 hKv1.1 (RCK1, RBK1,LO2750 MBK1, MK1, HuK1) KCNA2 hKv1.2 (RBK2, RBK5, NGK1, IIuKIV) KCNA3Kv1.3 (KV3, RGK5, HuKiIII, HPCN3) KCNA4 Kv1.4 (RCK4, RHK1, HuKII) KCNA5Kv1.5 (KV1, HPCN1, HK2) KCNA6 Kv1.6 (KV2, RCK2, IIBK2) KCNA7 Kv1.7 (MK6,RK6, US Pat. No. 5,559,009 HaK6) Kv2 (Shab) KCNB1 Kv2.1 (DRK1, mShab)M64228 KCNB2 Kv2.2 (CDRK1) US Pat. No. 5,710,019 K channel 2 Kv3 (Shaw)KCNB1 Kv3.1 (NGK2) KCNB2 Kv3.2 (KshIIIA) KCNB3 Kv3.3 (KshIIID) X607796KCNB4 Kv3.4 (Raw3) Kv4 (Sh1) KCND1 Kv4.1 (mShal, M64226 KShIVA) KCND2Kv4.2 (RK5, Rat Shal1) KCND3 Kv4.3 (KShIVB) WO 99/41372 hKv5.1 (IK8)Kv6.1 (K13) Kv7 Kv8.1 Kv9 Delayed Rectifier KvLQT1 AF000571 US Pat. No.5,599,673 HERG (crg) U04270 WO 99/20760 Calcium regulated Calciumregulated Big BKCa(hSLO) U11717 HBKb3 (β subunit) WO 99/42575 Maxi-K USPat. No. 5,776,734 US Pat. No. 5,637,470 Calcium regulated Calciumregulated Small KCNN1 SKCa1 U69883 KCNN2 SKCa2 U69882 KCNN3 SKCa3 U69884KCNN4 SKCa4 (IKCa1) Muscle Nerve 1999 22(6) 742-50 TWIK1 U33632Calcium Channels

Calcium channels are generally found in many cells where, among otherfunctions, they play important roles in signal transduction. Inexcitable cells, intracellular calcium supplies a maintained inwardcurrent for long depolarizing responses and serves as the link betweendepolarization and other intracellular signal transduction mechanisms.Like voltage-gated sodium channels, voltage-gated calcium channels havemultiple resting, activated, and inactivated states.

Multiple types of calcium channels have been identified in mammaliancells from various tissues, including skeletal muscle, cardiac muscle,lung, smooth muscle and brain, [see, e.g., Bean, B. P. (1989) Ann. Rev.Physiol. 51:367-384 and Hess, P. (1990) Ann. Rev. Neurosci. 56:337]. Thedifferent types of calcium channels have been broadly categorized intofour classes, L-, T-, N-, and P-type, distinguished by current kinetics,holding potential sensitivity and sensitivity to calcium channelagonists and antagonists. Four subtypes of neuronal voltage-dependentcalcium channels have been proposed (Swandulla, D. et al., Trends inNeuroscience 14:46, 1991).

Chloride Channels

Chloride channels are found in the plasma membranes of virtually everycell in the body. Chloride channels mediate a variety of cellularfunctions including regulation of transmembrane potentials andabsorption and secretion of ions across epithelial membranes. Whenpresent in intracellular membranes of the Golgi apparatus and endocyticvesicles, chloride channels also regulate organelle pH. For a review,see Greger, R. (1988) Annu. Rev. Physiol. 50:111-122.

Three distinct classes of chloride channels are apparent based on theirtype of regulation and structural conformation, Table 3. The first classincludes the GABA and Glycine receptor super families, the second classincludes the CFTR (Cystic fibrosis Transmembrane Conductance Regulator)and the third class includes the voltage regulated chloride channels.

TABLE 3 Chloride Channel Sub-type Summary Channel Tissue Type Sub-typeDistribution Reference Ligand GABA_(A) Receptor CNS & PNS Synapse 21,189-274 gated family (1995) Glycine Receptor CNS &PNS Trends Neurosci.14 family 458-461 (1991) cAMP CRTR Epithelial cells Science 245, 1066-regulated 1073 (1989) Voltage CIC-1 Skeletal muscle Nature 354, 301-304regulated (1991) CIC-1 Ubiquitous Nature 356, 57-60 (1992) CIC-Ka KidneyJ. Biol. Chem. 268, 3821-3824 (1993) CIC-Kb Kidney PNAS 91, 6943-6947(1994) CIC-3 Broad, e.g. kidney & Neuron 12, 597-604 brain (1994) CIC-4Broad, e.g. kidney & Hum. Nol. Genet. 3, brain 547-552 (1994) CIC-5Mainly kidney J. Biol. Chem. 270, 31172-31177 91995) CIC-6 UbiquitousFEBS Lett. 377, 15- 20 (1995) CIC-7 Ubiquitous FEBS Lett. 377, 15- 20(1995)

In some embodiments, the polypeptide portion of a subjectlight-regulated polypeptide is a glycine receptor.

In some embodiments, the polypeptide portion of a subjectlight-regulated polypeptide is an acetylcholine receptor. In someembodiments, the polypeptide portion of a subject light-regulatedpolypeptide is a nicotinic acetylcholine receptor. In some embodiments,the polypeptide portion of a subject light-regulated polypeptide is amuscarinic acetylcholine receptor. In some embodiments, the polypeptideportion of a subject light-regulated polypeptide is an M1, M2, M3, M4,or M5 muscarinic acetylcholine receptor subtype.

Cells

The embodiments further provide a cell comprising a subjectlight-regulated polypeptide. A subject cell finds use in a variety ofapplications, e.g., screening applications, such as identification ofagents that modulate the activity of a receptor; and researchapplications such as examination of a physiological event. Where thecell is used in a screening assay, the cell can be referred to as a“test cell.”

In some embodiments, the cell is a eukaryotic cell in in vitro cellculture, and is grown as an adherent monolayer, or in suspension. Inother embodiments, the cell is a eukaryotic cell and is part of a tissueor organ, either in vivo or in vitro. In other embodiments, the cell isa eukaryotic cell and is part of a living multicellular organism, e.g.,a protozoan, an amphibian, a reptile, a plant, an avian organism, amammal, a fungus, an algae, a yeast, a marine microorganism, a marineinvertebrate, an arthropod, an isopod, an insect, an arachnid, etc. Inother embodiments, the cell is a prokaryotic cell.

In other embodiments, the cell is a member of archaea, e.g., anarchaebacterium. Archaebacteria include a methanogen, an extremehalophile, an extreme thermophile, and the like. Suitable archaebacteriainclude, but are not limited to, any member of the groups Crenarchaeota(e.g., Sulfolobus solfataricus, Defulfurococcus mobilis, Pyrodictiumoccultum, Thermofilum pendens, Thermoproteus tenax), Euryarchacota(e.g., Thermococcus celer, Methanococcus thermolithotrophicus,Methanococcus jannaschii, Methanobacterium thermoautotrophicum,Methanobacterium formicicum, Methanothennus fervidus, Archaeoglobusfulgidus, Thermoplasma acidophilum, Haloferax volcanni, Methanosarcinabarkeri, Methanosaeta concilli. Methanospririllum hungatei,Methanomicrobium mobile), and Korarchaeota.

In some embodiments, the cell is of a particular tissue or cell type.For example, where the organism is a plant, the cell is part of thexylem, the phloem, the cambium layer, leaves, roots, etc. Where theorganism is an animal, the cell will in some embodiments be from aparticular tissue (e.g., lung, liver, heart, kidney, brain, spleen,skin, fetal tissue, etc.), or a particular cell type (e.g., neuronalcells, epithelial cells, endothelial cells, astrocytes, macrophages,glial cells, islet cells, T lymphocytes, B lymphocytes, etc.).

A subject cell is in many embodiments a unicellular organism, or isgrown in culture as a single cell suspension, or as monolayer. In someembodiments, a subject cell is a eukaryotic cell. Suitable eukaryoticcells include, but are not limited to, yeast cells, insect cells, plantcells, fungal cells, mammalian cells, and algal cells. Suitableeukaryotic host cells include, but are not limited to, Pichia pastoris,Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichiamembranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichiamethanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candidaalbicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusariumgramineurn, Fusarium venenaturn, Neurospora crassa, Chlarnydomonasreinhardtii, and the like.

Suitable mammalian cells include primary cells and immortalized celllines. Suitable mammalian cell lines include human cell lines, non-humanprimate cell lines, rodent (e.g., mouse, rat) cell lines, and the like.Suitable mammalian cell lines include, but are not limited to, HeLacells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHOcells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCCNo. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658),IIuh-7 cells, BIIK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No.CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse Lcells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No.CRL1573), HLHepG2 cells, and the like.

In some embodiments, the cell is a neuronal cell or a neuronal-likecell. The cells can be of human, non-human primate, mouse, or ratorigin, or derived from a mammal other than a human, non-human primate,rat, or mouse. In some embodiments, the neuronal cell is a primary cellisolated from an animal. In some embodiments, the neuronal cell orneuronal-liked cell is an immortalized cell line. Suitable cell linesinclude, but are not limited to, a human glioma cell line, e.g., SVGp12(ATCC CRL-8621), CCF-STTG1 (ATCC CRL-1718), SW 1088 (ATCC HTB-12), SW1783 (ATCC HTB-13), LLN-18 (ATCC CRL-2610), LNZTA3WT4 (ATCC CRL-11543),LNZTA3WT11 (ATCC CRL-11544), U-138 MG (ATCC HTB-16), U-87 MG (ATCCHTB-14), H4 (ATCC HTB-148), and LN-229 (ATCC CRL-2611); a humanmedulloblastoma-derived cell line, e.g., D342 Med (ATCC HTB-187), Daoy(ATCC HTB-186), D283 Med (ATCC HTB-185); a human tumor-derivedneuronal-like cell, e.g., PFSK-1 (ATCC CRL-2060), SK-N-DZ(ATCCCRL-2149), SK-N-AS (ATCC CRL-2137), SK-N-FI (ATCC CRL-2142), IMR-32(ATCC CCL-127), etc.; a mouse neuronal cell line, e.g., BC3H1 (ATCCCRL-1443), EOC1 (ATCC CRL-2467), C8-D30 (ATCC CRL-2534), C8-S (ATCCCRL-2535), Neuro-2a (ATCC CCL-131), NB41A3 (ATCC CCL-147), SW10 (ATCCCRL-2766), NG108-15 (ATCC HB-12317); a rat neuronal cell line, e.g.,PC-12 (ATCC CRL-1721). CTX TNA2 (ATCC CRL-2006), C6 (ATCC CCL-107), F98(ATCC CRL-2397), RG2 (ATCC CRL-2433), B35 (ATCC CRL-2754), R3 (ATCCCRL-2764), SCP (ATCC CRL-1700), OA1 (ATCC CRL-6538).

In other embodiments, the host cell is a plant cell. Plant cells includecells of monocotyledons (“monocots”) and dicotyledons (“dicots”).Guidance with respect to plant tissue culture may be found in, forexample: Plant Cell and Tissue Culture, 1994, Vasil and Thorpe Eds.,Kluwer Academic Publishers; and in: Plant Cell Culture Protocols(Methods in Molecular Biology 111), 1999, Hall Eds, Humana Press.

Suitable prokaryotic cells include bacteria (e.g., Eubacteria) andarchaebacteria. Suitable archaebacteria include a methanogen, an extremehalophile, an extreme thermophile, and the like. Suitable archaebacteriainclude, but are not limited to, any member of the groups Crenarchaeota(e.g., Sulfolobus solfataricus, Defillfurococcus mobilis, Pyrodictiumoccultuin, Therinofilum pendens, Thermoproteus tenax), Euryarchaeota(e.g., Thermococcus celer, Methanococcus thermolithotrophicus,Methanococcus jannaschii, Methanobacterium thermoautotrophicum,Methanobacterium formicicum, Methanothennus fervidus, Archaeoglobusfidgidus, Thermoplasma acidophilum, Haloferax volcanni, Methanosarcinabarkeri, Methanosaeta concilli, Methanospririllum hungatei,Methanotnicrobium mobile), and Korarchaeota. Suitable eubacteriainclude, but are not limited to, any member of Hydrogenobacteria,Thermotogales, Green nonsulfphur bacteria, Denococcus Group,Cyanobacteria, Purple bacteria, Planctomyces, Spirochetes, Green Sulphurbacteria, Cytophagas, and Gram positive bacteria (e.g., Mycobacteriumsp., Micrococcus sp., Streptomyces sp., Lactobacillus sp.,Helicobacterium sp., Clostridium sp., Mycoplasma sp., Bacillus sp.,etc.).

Suitable prokaryotic cells include, but are not limited to, any of avariety of laboratory strains of Escherichia coli, Lactobacillus sp.,Salmonella sp., Shigella sp., and the like. See, e.g., Carrier et al.(1992) J. Immunol. 148:1176-1181; U.S. Pat. No. 6,447,784; and Sizemoreet al. (1995) Science 270:299-302. Examples of Salmonella strains whichcan be employed in the embodiments include, but are not limited to,Salmonella typhi and S. typhimurium. Suitable Shigella strains include,but are not limited to, Shigella flexneri, Shigella sonnei, and Shigelladisenteriae. Typically, the laboratory strain is one that isnon-pathogenic. Non-limiting examples of other suitable bacteriainclude, but are not limited to, Bacillus subtilis, Pseudomonas pudita,Pseudomonas aeruginosa, Pseudomonas Rhodobacter sphaeroides, Rhodobactercapsulatus, Rhodospirillum rubrum, Rhodococcus sp., and the like. Insome embodiments, the cell is Escherichia coli.

Membranes

The embodiments further provide a membrane comprising a subjectlight-regulated polypeptide. In some embodiments, the membrane is abiological membrane (e.g., a lipid bilayer surrounding a biologicalcompartment such as a cell, including artificial cells, or a membranevesicle or sheet). In some embodiments, the membrane is part of a livingcell, as described above. In other embodiments, the membrane is anartificial (synthetic) membrane, e.g., a planar membrane, a liposome,etc.

In some embodiments, the artificial membrane is a lipid bilayer. Inother embodiments, the artificial membrane is a lipid monolayer. In someembodiments, the artificial membrane is part of a liposome. Liposomesinclude unilamellar vesicles composed of a single membrane or lipidbilayer, and multilamellar vesicles (MLVs) composed of many concentricmembranes (or lipid bilayers).

Artificial membranes, and methods of making same, have been described inthe art. See, e.g., U.S. Pat. No. 6,861,260; Kansy et al. (1998) J. Med.Chem. 41(7):1007-10; and Yang et al. (1996) Advanced Drug DeliveryReviews 23:229-256.

A subject artificial membrane will in some embodiments, include one ormore phospholipids. In some embodiments, the artificial membranecomprises a mixture of phospholipids containing saturated or unsaturatedmono or disubstituted fatty acids and a combination thereof. Thesephospholipids are in some embodiments selected fromdioleoylphosphatidylcholine, dioleoylphosphatidylserine,dioleoylphosphatidylethanolamine, dioleoylphosphatidylglycerol,dioleoylphosphatidic acid, palmitoyloleoylphosphatidylcholine, palmitoyloleoylphosphatidylserine, palmitoyloleoylphosphatidylethanolamine,palmitoyloleoylphophatidylglycerol, palmitoyloleoylphosphatidic acid,palmitelaidoyloleoylphosphatidylcholine,palmitelaidoyloleoylphosphatidylserine,palmitelaidoyloleoylphosphatidylethanolamine,palmitelaidoyloleoylphosphatidylglycerol,palmitelaidoyloleoylphosphatidic acid,myristoleoyloleoylphosphatidylcholine,myristoleoyloleoylphosphatidylserine,myristoleoyloleoylphosphatidylethanoamine,myristoleoyloleoylphosphatidylglycerol, myristoleoyloleoylphosphatidicacid, dilinoleoylphosphatidylcholine, dilinoleoylphosphatidylserine,dilinoleoylphosphatidylethanolamine, dilinoleoylphosphatidylglycerol,dilinoleoylphosphatidic acid, palmiticlinoleoylphosphatidylcholine,palmiticlinoleoylphosphatidylserine,palmiticlinoleoylphosphatidylethanolamine,palmiticlinoleoylphosphatidylglycerol, and palmiticlinoleoylphosphatidicacid. Suitable phospholipids also include the monoacylated derivativesof phosphatidylcholine (lysophophatidylidylcholine), phosphatidylserine(lysophosphatidylserine), phosphatidylethanolamine(lysophosphatidylethanolamine), phophatidylglycerol(lysophosphatidylglycerol) and phosphatidic acid (lysophosphatidicacid). The monoacyl chain in such lysophosphatidyl derivatives will insome embodiments be palimtoyl, oleoyl, palmitoleoyl, linoleoyl myristoylor myristoleoyl.

Methods of Modulating Protein Activity

The embodiments provide methods of modulating protein activity. Incertain aspects, the embodiments provide methods of modulating activityof a subject light-regulated polypeptide, where the method generallyinvolves changing the wavelength of light to which the light-regulatedpolypeptide is exposed. In certain aspects, the embodiments providemethods of modulating activity of a polypeptide, where the methodgenerally involves: a) contacting the polypeptide with a subjectsynthetic regulator, where the synthetic regulator binds to thepolypeptide, thereby generating a light-regulated polypeptide; and b)changing the wavelength of light to which the light-regulatedpolypeptide is exposed. In certain aspects, the present disclosureprovides methods of modulating activity of a ligand-binding polypeptide,where the method generally involves: a) contacting the ligand-bindingpolypeptide with a subject synthetic regulator, thereby generating alight-regulated polypeptide; and b) changing the wavelength of light towhich the light-regulated polypeptide is exposed. In other embodiments,as described below, in other aspects, the present disclosure providesmethods of modulating activity of a non-light-regulated polypeptidewhose activity is modulated by modulating the activity of alight-regulated polypeptide. In some aspects, the present disclosureprovides methods of modulating the activity of a non light-regulatedpolypeptide in a cell. The methods generally involve modulating anactivity of a light-regulated polypeptide in the cell, where modulationof the activity of the light-regulated polypeptide in the cell modulatesthe activity of the non-light-regulated polypeptide. Modulating theactivity of the light-regulated polypeptide results in modulation of thenon-light-regulated polypeptide.

As noted above, a “change in the wavelength of light to which thelight-regulated polypeptide is exposed” includes: 1) a change from λ₁ toλ₂; 2) a change from λ₂ to λ₁; 3) a change from λ₁ to darkness (nolight); and 4) a change from darkness to 2. In certain aspects, theembodiments provides methods of modulating activity of a native(wild-type) polypeptide, where the method generally involves: a)contacting a polypeptide with a subject synthetic regulator, where thesubject synthetic regulator hinds to the polypeptide, forming asynthetic regulator/polypeptide complex; and h) changing the wavelengthof light to which the synthetic regulator/polypeptide complex isexposed. As noted above, a “change in the wavelength of light to whichthe light-regulated polypeptide is exposed” includes: 1) a change fromλ₁ to λ₂; 2) a change from λ₂ to λ₁; 3) a change from λ₁ to darkness (nolight); and 4) a change from darkness to λ₁. The syntheticregulator/polypeptide complex is also referred to as a “light-regulatedpolypeptide.”

In some embodiments, the receptor or the light-regulated polypeptide ispresent in a cell-free in vitro system, e.g, the receptor or thelight-regulated polypeptide is not associated with a cell. In otherembodiments, the receptor or the light-regulated polypeptide isassociated with a cell, e.g., the receptor or the light-regulatedpolypeptide is integrated into a cell membrane in a cell, the receptoror the light-regulated polypeptide is in the cytosol of a cell, thereceptor or the light-regulated polypeptide is in an intracellularorganelle, etc. In other embodiments, the receptor or thelight-regulated polypeptide is in a synthetic membrane, e.g., in aplanar synthetic membrane, in a liposome, in a membrane of an artificialcell, etc. In some embodiments, the cell-associated polypeptide or thecell-associated light-regulated polypeptide is in a cell in vitro, e.g.,in a cell in a monolayer, in a cell in suspension, in an in vitrotissue, etc. In other embodiments, the cell-associated polypeptide orthe cell-associated light-regulated polypeptide is in a cell in vivo,e.g., in a cell of an organism, e.g., a living organism.

In some embodiments, the change in wavelength (from λ₁ to λ₂; from lightto darkness; or from darkness to light) results in a change in bindingof the ligand to a ligand-binding site. As used herein, a “change inbinding of a ligand to a ligand-binding site” includes increased bindingand decreased binding. As used herein, “increased binding” includes oneor more of: an increased probability of binding of the ligand to theligand-binding site; an increased binding affinity of the ligand for theligand-binding site; an increased local concentration of the ligand atthe ligand-binding site; and an increased occupancy of the ligand in theligand-binding site. As used herein, “decreased binding” includes one ormore of: a decreased probability of binding of the ligand to theligand-binding site; a decreased binding affinity of the ligand for theligand-binding site; a decreased local concentration of the ligand atthe ligand-binding site; and a decreased occupancy of the ligand in theligand-binding site. As used herein, the term “change in wavelength” towhich a synthetic regulator is exposed, or to which apolypeptide/synthetic light regulator complex is exposed, refers to achange in wavelength from λ₁ to λ₂; a change from light to darkness; ora change from darkness to light. An increase in binding includes anincrease of from about 10% to about 50%, from about 50% to about 2-fold,from about 2-fold to about 5-fold, from about 5-fold to about 10-fold,from about 10-fold to about 50-fold, from about 50-fold to about10²-fold, from about 10²-fold to about 10⁴-fold, from about 10⁴-fold toabout 10⁶-fold, from about 10⁶-fold to about 10⁸-fold, or a greater than10⁸-fold increase in binding. A decrease in binding includes a decreaseof from about 5% to about 10% to about 20% to about 30%, from about 30%to about 40%, from about 40% to about 50%, from about 50% to about 60%,from about 60% to about 70%, from about 70% to about 80%, from about 80%to about 90%, or from about 90% to 100% decrease in binding.

For example, in some embodiments, the ligand has a first probability ofbinding to the ligand site at a first wavelength of light; the ligandhas a second probability of binding to the ligand binding site at asecond wavelength of light; and the second probability is lower than thefirst probability. In other embodiments, the ligand has a firstprobability of binding to the ligand site at a first wavelength oflight; the ligand has a second probability of binding to the ligandbinding site at a second wavelength of light; and the second probabilityis higher than the first probability. In other embodiments, ligand has afirst probability of binding to the ligand site when exposed to light;the ligand has a second probability of binding to the ligand bindingsite in the absence of light (e.g., in darkness); and the secondprobability is lower than the first probability. In other embodiments,the ligand has a first probability of binding to the ligand site whenexposed to light; the ligand has a second probability of binding to theligand binding site in the absence of light and the second probabilityis higher than the first probability.

A change in wavelength can result in a change in activity of thelight-regulated protein. “Activity” will depend, in part, on thepolypeptide, and can include activity of an ion channel; activity of areceptor in transmitting a signal; etc.

In some embodiments, the change in wavelength results in binding of theligand to the ligand-binding site of the polypeptide. In someembodiments, the change in wavelength results in increased bindingaffinity of the ligand to the ligand-binding site for the polypeptide.In these embodiments, where the ligand is an agonist, and the changeresults in activation of the polypeptide; and where the ligand is anantagonist, the change results in block of activation of thepolypeptide; and where the ligand is an active site or pore blocker, thechange results in inhibition of the polypeptide; and where the ligand isa blocker of a site of interaction with other macromolecules, the changeinterferes with that interaction. In some embodiments, prolonged bindingof an agonist to the ligand-binding site results in desensitization orinactivation of the polypeptide. In other embodiments, binding of anantagonist blocks activation of the receptor.

In other embodiments, the change in wavelength results in lack ofbinding of the ligand to the ligand-binding site, e.g., removal of theligand from the ligand-binding site of the polypeptide. In otherembodiments, the change in wavelength results in reduced bindingaffinity of the ligand for the ligand-binding site, e.g., reducedbinding affinity of ligand for the ligand-binding site of thepolypeptide. In these embodiments, where the ligand is an antagonist,the change results in activation of said polypeptide; and where theligand is an agonist, the change results in deactivation oflight-regulated polypeptide, or recovery from desensitization orinactivation.

In some embodiments, the polypeptide/synthetic regulator complex isexposed to light of a first wavelength, where exposure to light of thefirst wavelength (λ₁) results in binding of the ligand to theligand-binding site (or increased binding affinity of the ligand for theligand-binding site); and the polypeptide/synthetic regulator complex issubsequently exposed to light of a second wavelength (λ₂), whereexposure to light of the second wavelength results in removal of theligand from the ligand-binding site (or reduced binding affinity of theligand for the ligand-binding site). This change in wavelength from afirst wavelength to a second wavelength (Δλ) can be repeated numeroustimes, such that the light is switched back and forth between λ₁ and λ₂.Switching between λ₁ and λ₂ results in switching or transition from aligand-bound state to a ligand-unbound state.

In some embodiments, the polypeptide/synthetic regulator complex isexposed to light of a first wavelength, where exposure to light of thefirst wavelength (λ₁) results in binding of the ligand to theligand-binding site (or increased binding affinity of the ligand for theligand-binding site); and the light is subsequently turned off, e.g.,the polypeptide/synthetic regulator complex is in darkness, wherekeeping the polypeptide/synthetic regulator complex in darkness resultsin removal of the ligand from the ligand-binding site (or reducedbinding affinity of the ligand for the ligand-binding site). This changefrom λ₁ to darkness can be reversed, e.g., from darkness to λ₁; andrepeated any number of times, as described above. In other embodiments,the polypeptide/synthetic regulator complex is exposed to light of afirst wavelength, where exposure to light of the first wavelength (λ₁)results in lack of binding of the ligand to the ligand-binding site (orreduced binding affinity of the ligand for the ligand-binding site); andthe light is subsequently turned off, e.g., the polypeptide/syntheticregulator complex is in darkness, where keeping thepolypeptide/synthetic regulator complex in darkness results in bindingof the ligand to the ligand-binding site (or increased binding affinityof the ligand for the ligand-binding site). This change from λ₁ todarkness can be reversed, e.g., from darkness to λ₁; and repeated anynumber of times, as described above.

As noted above, the change in wavelength can be repeated any number oftimes, e.g, from λ₁ to λ₂ and from λ₂ to λ₁; or from λ₁ to darkness andfrom darkness to λ₁. Thus, a subject method provides for inducing atransition or switch from a ligand-bound state of a protein to aligand-unbound state of the protein, or from a high affinity state to alow affinity state. Depending on whether the ligand is an agonist or anantagonist, the light-regulated polypeptide will in some embodiments beswitched from an active state to an inactive (or deactivated) state, orfrom an inactive (or deactivated) state to an active state.

The wavelength of light to which the light-regulated polypeptide isexposed ranges from 10⁸ m to about 1 in, e.g., from about 10⁻⁸ m toabout 10⁻⁷ m, from about 10⁻⁷ in to about 10⁻⁶ m, from about 10⁻⁶ m toabout 10⁻⁴ m, from about 10⁻⁴ m to about 10⁻² m, or from about 10⁻² m toabout 1 m. “Light,” as used herein, refers to electromagnetic radiation,including, but not limited to, ultraviolet light, visible light,infrared, and microwave.

The wavelength of light to which the light-regulated polypeptide isexposed ranges in some embodiments from about 200 nm to about 800 nm,e.g., from about 200 nm to about 250 nm, from about 250 nm to about 300nm, from about 300 nm to about 350 nm, from about 350 nm to about 400mil, from about 400 nm to about 450 nm, from about 450 nm to about 500nm, from about 500 nm to about 550 nm, from about 550 nm to about 600nm, from about 600 nm to about 650 nm, from about 650 nm to about 700nm, from about 700 nm to about 750 nm, or from about 750 nm to about 800nm, or greater than 800 nm.

In other embodiments, the wavelength of light to which thelight-regulated polypeptide is exposed ranges from about 800 nm to about2500 nm, e.g., from about 800 nm to about 900 nm, from about 900 nm toabout 1000 nm, from about 1000 nm to about 1200 nm, from about 1200 nmto about 1400 nm, from about 1400 nm to about 1600 nm, from about 1600nm to about 1800 nm, from about 1800 nm to about 2000 nm, from about2000 nm to about 2250 nm, or from about 2250 nm to about 2500 nm. Inother embodiments, the wavelength of light to which the light-regulatedpolypeptide is exposed ranges from about 2 nm to about 200 nm, e.g.,from about 2 nm to about 5 nm, from about 5 nm to about 10 nm, fromabout 10 nm to about 25 nm, from about 25 nm to about 50 nm, from about50 nm to about 75 nm, from about 100 nm, from about 100 nm to about 150nm, or from about 150 nm to about 200 nm.

The difference between the first wavelength and the second wavelengthcan range from about 10 nm to about 800 nm or more, e.g., from about 10nm to about 25 nm, from about 25 nm to about 50 nm, from about 50 nm toabout 100 nm, from about 100 nm to about 200 nm, from about 200 nm toabout 250 nm, from about 250 nm to about 500 nm, or from about 500 nm toabout 800 nm. Of course, where the light-regulated polypeptide isswitched from darkness to light, the difference in wavelength is fromessentially zero to a second wavelength.

The intensity of the light can vary from about 1 W/m² to about 50 W/m²,e.g., from about 1 W/m² to about 5 W/m², from about 5 W/m² to about 10W/m², from about 10 W/m², from about 10 W/m² to about 15 W/m², fromabout 15 W/m² to about 20 W/m², from about 20 W/m² to about 30 W/m²,from about 30 W/m² to about 40 W/m², or from about 40 W/m² to about 50W/m². The intensity of the light can vary from about 1 μW/cm² to about100 μW/cm², e.g., from about 1 μW/cm² to about 5 μW/cm², from about 5μW/cm² to about 10 μW/cm², from about 10 μW/cm² to about 20 μW/cm², fromabout 20 μW/cm² to about 25 μW/cm², from about 25 μW/cm² to about 50μW/cm², from about 50 μW/cm² to about 75 μW/cm², or from about 75 μW/cm²to about 100 μW/cm². In some embodiments, the intensity of light variesfrom about 1 μW/mm² to about 1 W/mm², e.g., from about 1 μW/mm² to about50 μW/mm², from about 50 μW/mm² to about 100 μW/mm², from about 100μW/mm² to about 500 μW/mm², from about 500 μW/mm² to about 1 mW/mm²,from about 1 mW/mm² to about 250 mW/mm², from about 250 mW/mm² to about500 mW/mm², or from about 500 mW/mm² to about 1 W/mm².

In some embodiments, the light-regulated polypeptide is regulated usingsound, instead of electromagnetic (EM) radiation (light). For example,in some embodiments, the light-regulated polypeptide is regulated usingultrasound to effect a change from a first isomeric form to a secondisomeric form.

The duration of exposure of the light-regulated polypeptide to light canvary from about 1 μsecond (μs) to about 60 seconds (s) or more, e.g.,from about 1 μs to about 5 μs, from about 5 μs to about 10 μs, fromabout 10 μs to about 25 μs, from about 25 μs to about 50 μs, from about50 μs to about 100 μs, from about 100 μs to about 250 μs, from about 250μs to about 500 μs, from about 500 μs to about 1 millisecond (ms), fromabout 1 ms to about 10 ms, from about 10 ms to about 50 ms, from about50 ms to about 100 ms, from about 100 ms to about 500 ms, from about 500ms to about 1 second, from about 1 second to about 5 seconds, from about5 seconds to about 10 seconds, from about 10 seconds to about 15seconds, from about 15 seconds to about 30 seconds, from about 30seconds to about 45 seconds, or from about 45 seconds to about 60seconds, or more than 60 seconds. In some embodiments, the duration ofexposure of the light-regulated polypeptide to light varies from about60 seconds to about 10 hours, e.g., from about 60 seconds to about 15minutes, from about 15 minutes to about 30 minutes, from about 30minutes to about 60 minutes, from about 60 minutes to about 1 hour, fromabout 1 hour to about 4 hours, from about 4 hours to about 6 hours, fromabout 6 hours to about 8 hours, or from about 8 hours to about 10 hours,or longer.

The duration of binding of the ligand portion of the synthetic regulatorto the ligand-binding site can vary from less than one second to days.For example, in some embodiments, the duration of binding of the ligandportion of the synthetic regulator to the ligand-binding site variesfrom about 0.5 second to about 1 second, from about 1 second to about 5seconds, from about 5 seconds to about 15 seconds, from about 15 secondsto about 30 seconds, from about 30 seconds to about 60 seconds, fromabout 1 minute to about 5 minutes, from about 5 minutes to about 15minutes, from about 15 minutes to about 30 minutes, or from about 30minutes to about 60 minutes. In other embodiments, the duration ofbinding of the ligand portion of the synthetic regulator to theligand-binding site varies from about 60 minutes to about 2 hours, fromabout 2 hours to about 4 hours, from about 4 hours to about 8 hours,from about 8 hours to about 12 hours, from about 12 hours to about 18hours, from about 18 hours to about 24 hours, from about 24 hours toabout 36 hours, from about 36 hours to about 48 hours, from about 48hours to about 60 hours, from about 60 hours to about 72 hours, fromabout 3 days to about 4 days, from about 4 days to about 5 days, or fromabout 5 days to about 7 days, or longer.

As noted above, in some embodiments, a synthetic regulator is membraneimpermeant. In these embodiments, a physical, electrical, or chemicalstimulus can be applied to a cell to facilitate entry of the syntheticregulator into the cell. An example of this is depicted schematically inFIG. 8. In some embodiments, a subject method of modulating proteinactivity involves contacting a cell comprising a polypeptide, whoseactivity is to be modulated, with a subject membrane-impermeantsynthetic regulator and further applying a physical, chemical, orelectrical stimulus to the cell, to facilitate entry of themembrane-impermeant synthetic regulator into the cell. For example, asubject method can involve contacting a cell comprising a polypeptide,whose activity is to be modulated, with a subject membrane-impermeantsynthetic regulator and an agonist of a nonselective ion channel, wherethe agonist facilitates entry of the membrane-impermeant syntheticregulator into the cell via the nonselective ion channel. Nonselectiveion channels include, e.g., ligand-gated nonselective cation channels.Nonselective cation channels include, e.g., TRPV₁, P2X₇R, and the like.P2X₇R (or P2X purinoceptor 7) is described in, e.g., Chessell et al.(2005) Pain 114:386; and Rassendren et al. (1997) J. Biol. Chem.272:5482. P2X₇R can be activated by adenosine triphosphate (ATP), or anATP analog. An example of a membrane-impermeant synthetic regulator isQAQ.

TRPV₁ (transient receptor potential cation channel, subfamily V, member1; also known as vanilloid receptor type 1), is a ligand-gatednonselective cation channel that is activated by a variety of endogenousand exogenous physical and chemical stimuli, including, e.g., heat over43° C., low pH, the endocannabinoid anandamide, N-arachidonoyl-dopamine,and capsaicin. For TRPV₁, see, e.g., Cui et al. (2006) J. Neurosci.26:9385.

TRPV₁ agonists include, e.g., capsaicin; a capsaicinoid (wherecapsaicinoids include, e.g., capsiate (4-hydroxy-3-methoxybenzyl(E)-8-methyl-6-nonenoate); dihydrocapsiate (4-hydroxy-3-methoxybenzyl8-methylnonanoate); nordihydrocapsiate (4-hydroxy-3-methoxybenzyl7-methyl-octanoate); capsiate derivatives such as vanillyl decanoate,vanillyl nonanoate, vanillyl octanoate and the like; fatty acid estersof vanillyl alcohol; and various straight chain or branched chain fattyacids which have a fatty acid chain length similar to that ofnordihydrocapsiate); resinifcratoxin; olvanil; tinyatoxin; a compound asdescribed in U.S. Patent Publication No. 2006/0240097; a compound asdescribed in U.S. Patent Publication No. 2009/0203774; a pentadienamidederivative as described in U.S. Patent Publication No. 2009/0203667; acompound as described in U.S. Patent Publication No. 2009/0170942; andthe like.

Modulating Activity of a Second, Non-Light-Regulated Polypeptide

In some embodiments, modulating the activity of a light-regulatedpolypeptide results in modulating the activity of a polypeptide otherthan the light-regulated polypeptide. Thus, in other aspects, thepresent disclosure provides methods of modulating activity of apolypeptide whose activity is modulated by modulating the activity of alight-regulated polypeptide. In some aspects, the present disclosureprovides methods of modulating the activity of a non light-regulatedpolypeptide in a cell. The methods generally involve modulating anactivity of a light-regulated polypeptide in the cell, where modulationof the activity of the light-regulated polypeptide in the cell modulatesthe activity of the non-light-regulated polypeptide.

A non-light-regulated polypeptide whose activity is modulated bymodulating the activity of a light-regulated polypeptide includes apolypeptide whose activity is modulated by a change in voltage of abiological membrane, a polypeptide whose activity is modulated bydepolarization of a biological membrane; a polypeptide whose activity ismodulated by a change in intracellular concentration of an ion (e.g., amonovalent or divalent ion, e.g., a monovalent or divalent cation); apolypeptide whose activity is modulated by phosphorylation; and thelike. As one non-limiting example, a light-regulated polypeptidecomprises a glutamate receptor (ligand-gated ion channel) as theligand-binding polypeptide, where the light-regulated polypeptide is inthe plasma membrane of a cell. Light activation of the light-regulatedglutamate receptor in the cell opens the channel, resulting in influx ofion and depolarization of the plasma membrane. Depolarization of theplasma membrane activates a voltage-gated ion channel, such as a calciumchannel Activation of the calcium channels is readily detected bystandard methods, e.g., use of an indicator dye, etc.).

As another non-limiting example, the light-regulated polypeptidecomprises a GPCR as the ligand-binding polypeptide. Activation of thelight-regulated GPCR activates an ion channel or an enzyme. Activationof the ion channel or enzyme is readily detected using standard methods,e.g., use of an indicator dye for the permeating ion, or a colorimetric,fluorimetric, or luminescence assay for the product of the enzyme. Asanother non-limiting example, the light-regulated polypeptide comprisesa receptor tyrosine kinase (RTK); and activation of the light-regulatedRTK results in phosphorylation of a downstream protein, e.g., atranscription factor. Activation of the transcription factor is readilydetected by, e.g., detecting a transcript. As another non-limitingexample, the light-regulated polypeptide comprises an opioid receptor.Modulation of the opioid receptor by exposure to light (or removal oflight) can modulate a potassium ion channel; and modulation of apotassium ion channel is readily detected using standard methods, e.g.,use of a dye for potassium ions.

As another non-limiting example, the non-light-regulated polypeptide isa voltage-dependent ion channel, and the light-regulated polypeptide isan ion channel (e.g., a ligand-gated ion channel); modulation of thelight-regulated ion channel by changing the wavelength of light to whichthe light-regulated ion channel is exposed leads to a change in themembrane potential of a cell harboring both the non-light-regulated,voltage-dependent ion channel and the light-regulated ion channel. Achange in the membrane potential of the cell modulates the activity ofthe non-light-regulated ion channel. Voltage-dependent (“voltage-gated”)ion channels include voltage-gated sodium channels, voltage-gatedpotassium channels, and voltage-gated calcium channels. Whether theactivity of the non-light-regulated, voltage-dependent ion channel ismodulated can be readily determined using any of a number of assaysdesigned to measure the intracellular concentration of an ion (e.g.,potassium, sodium, calcium), where such assays include use of dyes.

Utility

A subject synthetic regulator, a subject light-regulated polypeptide, asubject cell, and a subject method of modulating receptor function, areuseful in a wide variety of research applications, pharmaceuticalapplications, screening assays, therapeutic applications, and the like.

Research Applications

In some embodiments, a subject synthetic regulator or a subjectlight-regulated polypeptide is useful in studies of cell function, instudies of physiology of whole organisms, and the like.

In physiological studies, changing light exposure of a tissue, organ, orwhole organism (or a part of a whole organism) that includes a subjectlight-regulated protein provides a method of regulating a function inthe tissue, organ, or whole organism. For example, where thelight-regulated polypeptide is a light-regulated ligand-gated ionchannel, and the synthetic regulator comprises the ligand for theligand-gated ion channel, changing the wavelength of light to which thelight-regulated polypeptide is exposed will result in opening or closingof the ion channel, thereby altering ion concentration in cells in amanner that alters their activity (e.g., hormone or neurotransmittersecretion) or state (e.g., transcriptional or translational or metabolicstate) or electrical firing, etc.

Screening Methods

The embodiments provide methods of identifying an agent that modulates afunction (e.g., an activity) of a polypeptide. The methods generallyinvolve contacting a light-regulated polypeptide with a test agent; anddetermining the effect, if any, of the test agent on the activity of thelight-regulated polypeptide (or on the activity of a polypeptide that isregulated by the light-regulated polypeptide). The effect, if any, ofthe test agent on the activity of the light-regulated polypeptide isdetermined by exposing the light-regulated polypeptide to light of afirst wavelength. In the absence of the test agent, exposure of thelight-regulated polypeptide to light of a first wavelength induces atransition from a ligand-unbound state to a ligand-bound state. In thepresence of a test agent that affects binding of the ligand to theligand-binding site, the transition from the ligand-unbound state to aligand-bound state is inhibited.

In some embodiments, the light-regulated polypeptide is in vitro insolution (e.g., free of cells or membranes); and the assay is carriedout in vitro. In other embodiments, the light-regulated polypeptide isin a membrane (e.g., a synthetic membrane) in the absence of a livingcell (e.g., in a cell-free system); and the assay is carried out invitro. In other embodiments, the light-regulated polypeptide is in acell, e.g., a living cell in vitro or in vivo; and in some embodiments,the assay is carried out in vitro, and in other embodiments, the assayis carried out in vivo.

In some embodiments, the light-regulated polypeptide is in a cell (e.g.,is integrated into the plasma membrane, is in the cytosol of the cell,is in a subcellular organelle, is in the nucleus of the cell, or isintegrated into a membrane of a subcellular organelle). In theseembodiments, the cell comprising the light-regulated polypeptide is a“test cell.” The methods generally involve contacting the test cell witha test agent; and determining the effect, if any, of the test agent onthe activity of the light-regulated polypeptide.

In some aspects, the present disclosure provides methods for identifyingan agent that modulates a function (e.g., an activity) of anon-light-regulated polypeptide in the same solution, membrane, or cell,where the activity of the non-light-regulated polypeptide is modulatedby modulating the activity of a light-regulated polypeptide. The methodsgenerally involve contacting a non-light-regulated polypeptide and alight-regulated polypeptide (where the light-regulated polypeptide is ina solution, membrane, or cell) with a test agent; and determining theeffect, if any, of the test agent on the activity of thenon-light-regulated polypeptide (where the non-light regulatedpolypeptide is in the same solution, membrane, or cell as thelight-regulated polypeptide), where the activity of thenon-light-regulated polypeptide is modulated by changing the wavelengthof light to which the cell is exposed. Whether the activity of thenon-light regulated polypeptide is modulated is determined using anassay appropriate to the activity of the non-light-regulatedpolypeptide. For example, where the non-light-regulated polypeptide is acalcium channel, a calcium-sensitive dye, such as a Fura-2 dye, will insome embodiments be used to detect an effect of the test agent on theactivity of the calcium channel. For example, where thenon-light-regulated polypeptide is a sodium channel, a sodium-sensitivedye such as sodium-binding benzofuran isophthalate (SBFI) will in someembodiments be used to detect an effect of the test agent on theactivity of the sodium channel. Such an assay can be used, e.g., toidentify agents that modulate the activity of a voltage-dependent ionchannel where, e.g., the non-light regulated polypeptide is avoltage-dependent ion channel, and the light-regulated polypeptide is aligand-gated ion channel.

In some embodiments, the test agent is one that inhibits induction of atransition from a first, ligand-bound state to a second, ligand-unboundstate. For example, in some embodiments, a test agent of interest is onethat inhibits induction of a transition from a first, ligand-unboundstate to a second, ligand-bound state by at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 40%, at least about 50%, at leastabout 60%, at least about 70%, or at least about 80%, or more, comparedto the induction in the absence of the test agent.

The terms “candidate agent,” “test agent,” “agent,” “substance,” and“compound” are used interchangeably herein. Candidate agents encompassnumerous chemical classes, typically synthetic, semi-synthetic, ornaturally-occurring inorganic or organic molecules. Candidate agentsinclude those found in large libraries of synthetic or naturalcompounds. For example, synthetic compound libraries are commerciallyavailable from Maybridge Chemical Co. (Trevillet, Cornwall, UK),ComGenex (South San Francisco, Calif.), and MicroSource (New Milford,Conn.). A rare chemical library is available from Aldrich (Milwaukee,Wis.). Alternatively, libraries of natural compounds in the form ofbacterial, fungal, plant and animal extracts are available from Pan Labs(Bothell, Wash.) or are readily producible.

Candidate agents may be small organic or inorganic compounds having amolecular weight of more than 50 and less than about 2,500 daltons.Candidate agents may comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and mayinclude at least an amine, carbonyl, hydroxyl or carboxyl group, and maycontain at least two of the functional chemical groups. The candidateagents may comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Assays of the embodiments include controls, where suitable controlsinclude a sample (e.g., a sample comprising a subject polypeptide (asubject light-regulated polypeptide, e.g., a polypeptide in a complexwith a subject synthetic regulator) in the absence of the test agent).Generally a plurality of assay mixtures is run in parallel withdifferent agent concentrations to obtain a differential response to thevarious concentrations. Typically, one of these concentrations serves asa negative control, i.e. at zero concentration or below the level ofdetection.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc. that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used. Thecomponents of the assay mixture are added in any order that provides forthe requisite binding or other activity. Incubations are performed atany suitable temperature, e.g., between 4° C. and 40° C. Incubationperiods are selected for optimum activity, but may also be optimized tofacilitate rapid high-throughput screening. Typically between 0.1 hourand 1 hour will be sufficient.

The screening methods may be designed a number of different ways, wherea variety of assay configurations and protocols may be employed, as areknown in the art. The above components of the method may be combined atsubstantially the same time or at different times. In some embodiments,a subject method will include one or more washing steps.

In some embodiments, the light regulated polypeptide is assayed in amembrane-free, cell free assay. In other embodiments, the lightregulated polypeptide is integrated into an artificial membrane. Inother embodiments, light regulated polypeptide is integrated into abiological membrane. In other embodiments, the light regulatedpolypeptide is in a living cell, e.g., in the cytosol, in the nucleus,in an intracellular organelle, in the plasma membrane, or in anintracellular membrane of the cell.

Biological cells which are suitable for use in a subject screening assayinclude, but are not limited to, primary cultures of mammalian cells,transgenic (non-human) organisms and mammalian tissue. Cells inscreening assays may be dissociated either immediately or after primaryculture. Cell types include, but are not limited to white blood cells(e.g. leukocytes), hepatocytes, pancreatic beta-cells, neurons, smoothmuscle cells, intestinal epithelial cells, cardiac myocytes, glialcells, and the like.

Biological cells which are suitable for use in a subject screening assayinclude cultured cell lines (e.g., immortalized cell lines).Representative suitable cultured cell lines derived from humans andother mammals include, but are not limited to, HeLa cells (e.g.,American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g.,ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573),Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHKcells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells,COS-7 cells (ATCC No. CRL1651). RAT1 cells, mouse L cells (ATCC No.CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2cells, and the like.

In some embodiments, the cell is a neuronal cell or a neuronal-likecell. The cells can be of human, non-human primate, mouse, or ratorigin, or derived from a mammal other than a human, non-human primate,rat, or mouse. Suitable cell lines include, but are not limited to, ahuman glioma cell line, e.g., SVGp12 (ATCC CRL-8621), CCF-STTG1 (ATCCCRL-1718), SW 1088 (ATCC HTB-12), SW 1783 (ATCC HTB-13), LLN-18 (ATCCCRL-2610), LNZTA3WT4 (ATCC CRL-11543), LNZTA3WT11 (ATCC CRL-11544),U-138 MG (ATCC HTB-16), U-87 MG (ATCC HTB-14), H4 (ATCC HTB-148), andLN-229 (ATCC CRL-2611); a human medulloblastoma-derived cell line, e.g.,D342 Med (ATCC HTB-187), Daoy (ATCC HTB-186), D283 Med (ATCC HTB-185); ahuman tumor-derived neuronal-like cell, e.g., PFSK-1 (ATCC CRL-2060),SK-N-DZ (ATCCCRL-2149), SK-N-AS (ATCC CRL-2137), SK-N-FI (ATCCCRL-2142), IMR-32 (ATCC CCL-127), etc.; a mouse neuronal cell line,e.g., BC3H1 (ATCC CRL-1443), EOC1 (ATCC CRL-2467), C8-D30 (ATCCCRL-2534), C8-S (ATCC CRL-2535), Neuro-2a (ATCC CCL-131), NB41A3 (ATCCCCL-147), SW10 (ATCC CRL-2766), NG108-15 (ATCC HB-12317); a rat neuronalcell line, e.g., PC-12 (ATCC CRL-1721), CTX TNA2 (ATCC CRL-2006), C6(ATCC CCL-107), F98 (ATCC CRL-2397), RG2 (ATCC CRL-2433), B35 (ATCCCRL-2754), R3 (ATCC CRL-2764), SCP (ATCC CRL-1700), OA1 (ATCC CRL-6538).

In some embodiments, the readout for an effect on the activity of thelight regulated polypeptide is a direct measure of the activity of thelight regulated polypeptide. A direct effect on the light regulatedpolypeptide is detected using an assay appropriate to the particularpolypeptide. For example, where the light regulated receptor is an ionchannel, the effect, if any, of the test agent on the activity of theion channel is in some embodiments detected by detecting a change in theintracellular concentration of an ion. A change in the intracellularconcentration of an ion can be detected using an indicator appropriateto the ion whose influx is controlled by the channel. For example, wherethe ion channel is a potassium ion channel, a potassium-detecting dye isused; where the ion channel is a calcium ion channel, acalcium-detecting dye is used; etc.

Suitable voltage-sensitive dyes include, but are not limited to,merocyanine-oxazolone dyes (e.g., NK2367); merocyanine-rhodanine dyes(e.g., NK2495, NK2761, NK2776, NK3224, and NK3225); oxonol dyes (e.g.,RH155, RH479, RH482, RH1691, RH1692, and RH1838); styryl dyes (e.g.,RH237, RH414, RH421, RH437, RH461, RH795, JPW1063, JPW3028, di-4-ANEPPS,di-9-ANEPPS, di-2-ANEPEQ, di-12-ANEPEQ, di-8-ANEPPQ, and di-12-ANEPPQ);and the like.

Suitable intracellular K⁺ ion-detecting dyes include, but are notlimited to, K⁺-binding benzofuran isophthalate and the like.

Suitable intracellular Ca²⁺ ion-detecting dyes include, but are notlimited to, fura-2, bis-fura 2, indo-1, Quin-2, Quin-2 AM,Benzothiaza-1, Benzothiaza-2, indo-5F, Fura-FF, BTC, Mag-Fura-2,Mag-Fura-5, Mag-Indo-1, fluo-3, rhod-2, fura-4F, fura-5F, fura-6F,fluo-4, fluo-5F, fluo-5N, Oregon Green 488 BAPTA, Calcium Green,Calcein, Fura-C18, Calcium Green-C18, Calcium Orange, Calcium Crimson,Calcium Green-5N, Magnesium Green, Oregon Green 488 BAPTA-1, OregonGreen 488 BAPTA-2, X-rhod-1, Fura Red, Rhod-5F, Rhod-5N, X-Rhod-5N,Mag-Rhod-2, Mag-X-Rhod-1, Fluo-5N, Fluo-5F, Fluo-4FF, Mag-Fluo-4,Aequorin, dextran conjugates or any other derivatives of any of thesedyes, and others (see, e.g., the catalog or Internet site for MolecularProbes, Eugene, see, also, Nuccitelli, ed., Methods in Cell Biology,Volume 40: A Practical Guide to the Study of Calcium in Living Cells,Academic Press (1994); Lambert, ed., Calcium Signaling Protocols(Methods in Molecular Biology Volume 114), Humana Press (1999); W. T.Mason, ed., Fluorescent and Luminescent Probes for Biological Activity.A Practical Guide to Technology for Quantitative Real-Time Analysis,Second Ed, Academic Press (1999); Calcium Signaling Protocols (Methodsin Molecular Biology), 2005, D. G. Lamber, ed., Humana Press.).

In particular embodiments of interest, a subject screening method isuseful for identifying agents that alter the sense of taste. In otherembodiments, a subject screening method is useful for identifying agentsthat affect one or more neurological functions of a mammalian subject.In other embodiments, a subject screening method is useful foridentifying agents that are selective for a particular receptor type orsubtype, where the screening method involves determining the effect ofthe agent on a first subtype and a second subtype, where an effect onthe first subtype, and a reduced effect (or substantially no effect) onthe second subtype indicates selectivity of the test agent for the firstsubtype. In other embodiments, as noted above, a subject screeningmethod is useful for identifying agents that modulate the activity of avoltage-gated ion channel.

Therapeutic Applications

A subject synthetic regulator of protein function is suitable for use ina variety of therapeutic applications, which are also provided. In someembodiments, a subject synthetic regulator of protein function is usefulin restoring light sensitivity to a retina that has reduced lightsensitivity. In other embodiments, a subject synthetic regulator ofprotein function is useful as a local anesthetic. In other embodiments,a subject synthetic regulator is useful as an anti-convulsant, e.g., inthe treatment of epilepsy.

Restoring Light Sensitivity to a Retina

The embodiments provide a method for restoring light sensitivity to aretina, or conferring light sensitivity to a cell in the eye, the methodgenerally involving administering to an individual in need thereof aneffective amount of a subject synthetic regulator of protein functionlocally, e.g., in or around the eye.

A subject synthetic regulator that is suitable for this applicationcomprises a ligand that confers light sensitivity on one or more cellsin the eye, e.g., retinal pigment epithelial cells; and cells disposedin the neurosensory retina, for example, photoreceptor cells and Muellercells. A pharmaceutical composition comprising a subject syntheticregulator is administered in or around the eye; the synthetic regulatorattaches to a protein in a cell in the eye, and confers lightsensitivity to the cell. Suitable pharmaceutical compositions aredescribed in detail below. For example, the synthetic regulator canconfer light sensitivity on a retinal ganglion.

A pharmaceutical composition comprising a subject synthetic regulatorthat confers light sensitivity on a cell can be delivered to the eyethrough a variety of routes. A subject pharmaceutical composition may bedelivered intraocularly, by topical application to the eye or byintraocular injection into, for example the vitreous or subretinal(interphotoreceptor) space. Alternatively, a subject pharmaceuticalcomposition may be delivered locally by insertion or injection into thetissue surrounding the eye. A subject pharmaceutical composition may bedelivered systemically through an oral route or by subcutaneous,intravenous or intramuscular injection. Alternatively, a subjectpharmaceutical composition may be delivered by means of a catheter or bymeans of an implant, wherein such an implant is made of a porous,non-porous or gelatinous material, including membranes such as silasticmembranes or fibers, biodegradable polymers, or proteinaceous material.A subject pharmaceutical composition can be administered prior to theonset of the condition, to prevent its occurrence, for example, duringsurgery on the eye, or immediately after the onset of the pathologicalcondition or during the occurrence of an acute or protracted condition.

The effects of therapy for an ocular disorder as described herein can beassessed in a variety of ways, using methods known in the art. Forexample, the subject's vision can be tested according to conventionalmethods. Such conventional methods include, but are not necessarilylimited to, electroretinogram (ERG), focal ERG, tests for visual fields,tests for visual acuity, ocular coherence tomography (OCT), Fundusphotography, Visual Evoked Potentials (VEP) and Pupillometry. Ingeneral, the embodiments provide for maintenance of a subject's vision(e.g., prevention or inhibition of vision loss of further vision lossdue to photoreceptor degeneration), slowing progression of vision loss,or in some embodiments, providing for improved vision relative to thesubject's vision prior to therapy.

Exemplary conditions of particular interest which are amenable totreatment according to the methods of the embodiments include, but arenot necessarily limited to, diabetic retinopathy, age-related maculardegeneration (AMD or ARMD) (wet form); dry AMD; retinopathy ofprematurity; retinitis pigmentosa (RP); diabetic retinopathy; andglaucoma, including open-angle glaucoma (e.g., primary open-angleglaucoma), angle-closure glaucoma, and secondary glaucomas (e.g.,pigmentary glaucoma, pseudoexfoliative glaucoma, and glaucomas resultingfrom trauma and inflammatory diseases).

Further exemplary conditions amenable to treatment according to theembodiments include, but are not necessarily limited to, retinaldetachment, age-related or other maculopathies, photic retinopathies,surgery-induced retinopathies, toxic retinopathies, retinopathy ofprematurity, retinopathies due to trauma or penetrating lesions of theeye, inherited retinal degenerations, surgery-induced retinopathies,toxic retinopathies, retinopathies due to trauma or penetrating lesionsof the eye.

Specific exemplary inherited conditions of interest for treatmentaccording to the embodiments include, but are not necessarily limitedto, Bardet-Biedl syndrome (autosomal recessive); Congenital amaurosis(autosomal recessive); Cone or cone-rod dystrophy (autosomal dominantand X-linked forms); Congenital stationary night blindness (autosomaldominant, autosomal recessive and X-linked forms); Macular degeneration(autosomal dominant and autosomal recessive forms); Optic atrophy,autosomal dominant and X-linked forms); Retinitis pigmentosa (autosomaldominant, autosomal recessive and X-linked forms); Syndromic or systemicretinopathy (autosomal dominant, autosomal recessive and X-linkedforms); and Usher syndrome (autosomal recessive).

Local Anesthetic

The embodiments provide a method of reducing or preventing pain in anindividual, the method generally involving: a) administering to anindividual in need thereof an effective amount of a subject syntheticregulator of protein function, where the synthetic regulator of proteinfunction comprises a ligand that blocks a pain response or a painsignal, where the synthetic regulator binds to receptor or a channel,forming complex between the synthetic regulator and the receptor orchannel; and b) exposing the receptor/regulator complex orchannel/regulatory complex to a wavelength of light that provides forbinding of the ligand to the receptor or channel. For example, in someembodiments, the protein is a cation channel, and the syntheticregulator binds to the cation channel, forming a cationchannel/regulator complex, where the channel/regulator complex isexposed to a wavelength of light that provides for blocking of thechannel, e.g., a Na⁺ channel, an N-type Ca²⁺ channel, etc.

An “effective amount” of a subject synthetic regulator is an amount thatis effective to reduce pain by at least 30%, 40%, 60%, 70%, 80%, 90% or100% for a period of time of from about 15 minutes to 5 days, e.g., fromabout 15 minutes to about 30 minutes, from about 30 minutes to about 60minutes, from about 1 hour to about 4 hours, from about 4 hours to about8 hours, from about 8 hours to about 16 hours, from about 16 hours toabout 24 hours, from about 24 hours to about 36 hours, from about 36hours to about 48 hours, from about 48 hours to about 3 days, or fromabout 3 days to about 5 days. The effectiveness of a subject syntheticregulator in treating nociceptive pain can be determined by observingone or more clinical symptoms or physiological indicators associatedwith nociceptive pain.

In these embodiments, a suitable synthetic regulator includes one thatcomprises, as a ligand, an opioid analgesic. Suitable ligands include,but are not limited to, morphine, oxycodone, fentanyl, pentazocine,hydromorphone, meperidine, methadone, levorphanol, oxymorphone,levallorphan, codeine, dihydrocodeine, hydrocodone, propoxyphene,nalmefene, nalorphine, naloxone, naltrexone, buprenorphine, butorphanol,nalbuphine, and pentazocine. In other embodiments, a suitable syntheticregulator comprises a ligand moiety selected from lidocaine, novocaine,xylocalne, lignocaine, novocaine, carbocaine, etidocaine, tetracaine,procaine, prontocaine, prilocalne, bupivacaine, cinchocaine,mcpivacaine, quinidine, flecainide, procaine,N-[[2′-(aminosulfonyl)biphenyl-4-yl]methyl]-N′-(2,2′-bithien-5-ylmethyl)succinamide(BPBTS), QX-314, saxitoxin, tetrodotoxin, and a type III conotoxin.

As noted above, in some embodiments, a subject synthetic regulator ismembrane impermeant. Where a subject method involves administration of amembrane-impermeant synthetic regulator, a subject method can involveadministering a subject membrane-impermeant synthetic regulator to anindividual, and further applying a physical, chemical, or electricalstimulus to the individual, to facilitate entry of themembrane-impermeant synthetic regulator into a cell in the individual.For example, a subject method can involve administering a subjectmembrane-impermeant synthetic regulator and an agonist of a nonselectiveion channel, where the agonist facilitates entry of themembrane-impermeant synthetic regulator into a cell via the nonselectiveion channel. Nonselective ion channels include, e.g., ligand-gatednonselective cation channels. Suitable agonists of nonselective ionchannels include those described hereinabove. For example, TRPV₁agonists include, e.g., capsaicin; a capsaicinoid (where capsaicinoidsinclude, e.g., capsiate (4-hydroxy-3-methoxybenzyl(E)-8-methyl-6-nonenoate); dihydrocapsiate (4-hydroxy-3-methoxybenzyl8-methylnonanoate); nordihydrocapsiate (4-hydroxy-3-methoxybenzyl7-methyl-octanoate); capsiate derivatives such as vanillyl decanoate,vanillyl nonanoate, vanillyl octanoate and the like; fatty acid estersof vanillyl alcohol; and various straight chain or branched chain fattyacids which have a fatty acid chain length similar to that ofnordihydrocapsiate); resiniferatoxin; olvanil; tinyatoxin; a compound asdescribed in U.S. Patent Publication No. 2006/0240097; a compound asdescribed in U.S. Patent Publication No. 2009/0203774; a pentadienamidederivative as described in U.S. Patent Publication No. 2009/0203667; acompound as described in U.S. Patent Publication No. 2009/0170942; andthe like. Suitable P2X₇R agonists include ATP and ATP analogs thatfunction as P2X₇R agonists.

The present disclosure provides pharmaceutical compositions comprising asubject synthetic regulator. In some embodiments, a subjectpharmaceutical composition is suitable for administering to anindividual in need of a local anesthetic. Individuals in need of a localanesthetic include an individual who is about to undergo a surgicalprocedure, and an individual who has undergone a surgical procedurewithin the last 5 minutes to within the last 72 hours. Individuals inneed of a local anesthetic further include individuals having a wound,e.g., a superficial wound.

A pharmaceutical composition comprising a subject synthetic regulatormay be administered to a patient alone, or in combination with othersupplementary active agents. The pharmaceutical compositions may bemanufactured using any of a variety of processes, including, withoutlimitation, conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping, and lyophilizing.The pharmaceutical composition can take any of a variety of formsincluding, without limitation, a sterile solution, suspension, emulsion,lyophilisate, tablet, pill, pellet, capsule, powder, syrup, elixir orany other dosage form suitable for administration.

A pharmaceutical composition comprising a subject synthetic regulatorcan optionally include a pharmaceutically acceptable carrier(s) thatfacilitate processing of an active ingredient into pharmaceuticallyacceptable compositions. As used herein, the term “pharmacologicallyacceptable carrier” refers to any carrier that has substantially no longterm or permanent detrimental effect when administered and encompassesterms such as “pharmacologically acceptable vehicle, stabilizer,diluent, auxiliary or excipient.” Such a carrier generally is mixed withan active compound, or permitted to dilute or enclose the activecompound and can be a solid, semi-solid, or liquid agent. It isunderstood that the active ingredients can be soluble or can bedelivered as a suspension in the desired carrier or diluent. Any of avariety of pharmaceutically acceptable carriers can be used including,without limitation, aqueous media such as, e.g., distilled, deionizedwater, saline; solvents; dispersion media; coatings; antibacterial andantifungal agents; isotonic and absorption delaying agents; or any otherinactive ingredient. Selection of a pharmacologically acceptable carriercan depend on the mode of administration. Except insofar as anypharmacologically acceptable carrier is incompatible with the activeingredient, its use in pharmaceutically acceptable compositions iscontemplated. Non-limiting examples of specific uses of suchpharmaceutical carriers can be found in “Pharmaceutical Dosage Forms andDrug Delivery Systems” (Howard C. Ansel et al., eds., LippincottWilliams & Wilkins Publishers, 7^(th) ed. 1999); “Remington: The Scienceand Practice of Pharmacy” (Alfonso R. Gennaro ed., Lippincott, Williams& Wilkins, 20^(th) 2000); “Goodman & Gilman's The Pharmacological Basisof Therapeutics” Joel G. Hardman et al., eds., McGraw-Hill Professional,10.sup.th ed. 2001); and “Handbook of Pharmaceutical Excipients”(Raymond C. Rowe et al., APhA Publications, 4^(th) edition 2003).

A subject pharmaceutical composition can optionally include, withoutlimitation, other pharmaceutically acceptable components, including,without limitation, buffers, preservatives, tonicity adjusters, salts,antioxidants, physiological substances, pharmacological substances,bulking agents, emulsifying agents, wetting agents, sweetening orflavoring agents, and the like. Various buffers and means for adjustingpH can be used to prepare a pharmaceutical composition disclosed in thepresent specification, provided that the resulting preparation ispharmaceutically acceptable. Such buffers include, without limitation,acetate buffers, citrate buffers, phosphate buffers, neutral bufferedsaline, phosphate buffered saline and borate buffers. It is understoodthat acids or bases can be used to adjust the pH of a composition asneeded. Pharmaceutically acceptable antioxidants include, withoutlimitation, sodium metabisulfite, sodium thiosulfate, acetylcysteine,butylated hydroxyanisole and butylated hydroxytoluene. Usefulpreservatives include, without limitation, benzalkonium chloride,chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuricnitrate and a stabilized oxy chloro composition, for example, PURITE™.Tonicity adjustors suitable for inclusion in a subject pharmaceuticalcomposition include, without limitation, salts such as, e.g., sodiumchloride, potassium chloride, mannitol or glycerin and otherpharmaceutically acceptable tonicity adjustor. It is understood thatthese and other substances known in the art of pharmacology can beincluded in a subject pharmaceutical composition.

Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

Routes of administration suitable for the methods of the embodimentsinclude both systemic and local administration. In some embodiments, asubject pharmaceutical composition comprising a subject syntheticregulator is administered locally. As non-limiting examples, apharmaceutical composition useful for treating nociceptive pain can beadministered orally; by subcutaneous pump; by dermal patch; byintravenous, subcutaneous or intramuscular injection; by topical drops,creams, gels, sprays, or ointments; as an implanted or injected extendedrelease formulation; as a bioerodable or non-bioerodable deliverysystem; by subcutaneous minipump or other implanted device; byintrathecal pump or injection; or by epidural injection. In someembodiments, a subject pharmaceutical composition comprising a subjectsynthetic regulator is administered sublingually. In some embodiments, asubject pharmaceutical composition comprising a subject syntheticregulator is administered topically to gum tissue. In some embodiments,a subject pharmaceutical composition comprising a subject syntheticregulator is injected into gum tissue. In some embodiments, a subjectpharmaceutical composition comprising a subject synthetic regulator isadministered topically to the skin. In some embodiments, a subjectpharmaceutical composition comprising a subject synthetic regulator isadministered at or near a site of a surgical incision. In someembodiments, a subject pharmaceutical composition comprising a subjectsynthetic regulator is administered intramuscularly at the site of asurgical incision. For example, in some embodiments, a subjectpharmaceutical composition comprising a subject synthetic regulator isadministered at a surgical site, and before the surgical wound isclosed, the synthetic regulator/target protein complex is exposed tolight of a wavelength that induces binding of the ligand to the protein.In some embodiments, a subject pharmaceutical composition isadministered (e.g., injected) at or near a nerve. Thus, in someembodiments, a subject pharmaceutical composition is formulated forinjection at or near a nerve. For example, for oral surgery, a subjectpharmaceutical composition is injected at or near a nerve in gum tissue.

In some embodiments, a subject pharmaceutical composition comprising asubject synthetic regulator is administered just before surgery, e.g.,from about 1 minute to about 2 hours before surgery, e.g., from about 1minute to about 5 minutes, from about 5 minutes to about 15 minutes fromabout 15 minutes to about 30 minutes, from about 30

A subject synthetic regulator comprising a ligand that provides for painprevention is suitable for preventing or reducing pain in an individualin need thereof. Individuals in need of treatment with a subjectsynthetic regulator comprising a ligand that provides for painprevention include individuals who are about to undergo surgery, e.g.,individuals who are scheduled to undergo a surgical procedure in thenext 5 minutes to 72 hours; individuals who are undergoing a surgicalprocedure; and individuals who have undergone a surgical procedurewithin the previous 5 minutes to 1 hour. Thus, individuals sufferingfrom post-operative pain are suitable for treatment. A subject syntheticregulator comprising a ligand that provides for pain prevention is alsosuitable for preventing or reducing pain in an individual having awound, e.g., a superficial wound.

Anti-Convulsant Applications

In some embodiments, a subject synthetic regulator comprises, as aligand, a ligand for a sodium channel, a potassium channel, or a GABAreceptor, where the ligand functions as an anti-convulsant. In someembodiments, the synthetic regulator is administered in a pharmaceuticalcomposition, as described supra and infra.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the embodiments, and are not intended to limit the scope ofwhat the inventors regard as their invention nor are they intended torepresent that the experiments below are all or the only experimentsperformed. Efforts have been made to ensure accuracy with respect tonumbers used (e.g. amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is weight averagemolecular weight, temperature is in degrees Celsius, and pressure is ator near atmospheric. Standard abbreviations may be used, e.g., bp, basepair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min,minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp,base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p.,intraperitoneal(ly); s.c., subcutaneous(ly); and the like.

Example 1 Photochromic, Internal Blockers of Voltage-Gated PotassiumChannels Materials and Methods

Cell Culture, Plasmids and Transfection.

HEK293 cells were grown in DMEM containing 5% FBS. For HEK293T cells,500 mg/ml G-418 was also included. For electrophysiology studies, cellswere plated at 12×10³ cells/cm² on poly(L-lysine) coated coverslips andtransfected the cells using the calcium phosphate method. K+ channelcurrents were recorded 24-48 h after transfection. Hippocampal neuronswere prepared from neonatal rats according to standard procedures,plated at 50×10³ cells/cm² on poly(L-lysine) coated coverslips, andgrown in minimum essential medium containing 5% FBS, 20 mM glucose, B27(Invitrogen), glutamine and Mito+ Serum Extender (BD Biosciences).Currents were recorded 14-25 days (d) after plating. Animal care andexperimental protocols were approved by the University of CaliforniaBerkeley Animal Care and Use Committee.

Electrophysiology Recordings from Cultured Cells.

Currents were recorded in the whole-cell patch clamp configuration usingpipettes with 3-5 MSS resistance. To elicit voltage-gated K⁺ currentsfrom neurons and HEK293 cells, holding potential was set to −70 mV andstepped to +30 or +40 mV for 200, 250 or 300 ins. The bath solution forwhole cell recordings contained in mM: 138 NaCl, 1.5 KCl, 1.2 MgCl₂, 2.5CaCl₂, 5 HEPES, 10 glucose, pH 7.4 and during recordings from neurons,20 bicuculline, 25 μM 6,7-dinitroquinoxaline-2,3-[1H,4H]-dione (DNQX)and 1 μM tetradotoxm (TTX). The intracellular solution contained in mM:10 NaCl, 135 K-gluconate, 10 HEPES, 2 MgCl₂, 2 MgATP, EGTA, pH 7.4. Thebath solution for inside-out patch recordings contained in mM: 160 KCl,0.5 MgCl₂, 1 EGTA, 10 HEPES, pH 7.4. The pipette solution for inside-outpatch recordings contained in mM: 150 NaCl, 10 KCl, 10 HEPES, 1 MgCl₂, 3CaCl₂, pH 7.4. Solutions were adjusted to 300-310 mOsm.

AzoQA Treatment.

Cells were rinsed with whole cell bath solution and then incubated at37° C. in the dark for 15 minutes at the indicated concentrations ofAzoQA diluted from 200 mM DMSO stocks into bath solution and then rinsedwith bath solution prior to recording. During some experiments, AAQ waslocally perfused onto cells or patches at the indicated concentrationsin either whole cell or inside-out patch solution after recording hadbegun.

Illumination Conditions.

To achieve photoswitching in living cells, a xenon lamp (175 W) withnarrow band-pass filters (380BP10 and 500BP5) was used. Light output wasmeasured using a handheld Newport meter (840-C model). At the back ofthe objective, light output was 0.3 mW/cm² for 380-nm light and 2.5mW/cm² for 500-nm light. When measured through a 40× objective andnormalized to the focal area at the specimen plane, light output was 0.5mW/mm² and 3.5 mW/mm² for 380-nm and 500-nm light, respectively. Forsome experiments, we used a monochromator (Polychrome V; TILL Photonics)for illumination.

All data reported are averages±s.d.

Results

AAQ and EtAcAQ Effect

Acrylamide Azobenzene Quaternary ammonium (AAQ) 1, named for theacrylamide, azobenzene and quaternary ammonium components (FIG. 2a ),can be a useful tool due to its persistent effects and lack of toxicity.If ligand-binding were to direct the covalent reaction of AAQ withsurface residues near the external TEA-binding site of potassiumchannels, it can be possible to alter the rate of labeling by alteringthe TEA-binding site affinity with site-directed mutagenesis or bylabeling with AAQ in the presence of a ligand that competes for the samesite. Additionally, it can be possible to prevent covalent modificationaltogether by genetically removing the nucleophilic residue(s). Thesepossibilities were tested using whole-cell recordings from HEK 293 cellsexpressing Shaker IR, which lacks fast inactivation but retains slow orP/C-type inactivation.

Three Shaker mutants with external TEA affinities spanning 3 orders ofmagnitude all appeared to react at similar rates with extracellularlyapplied AAQ and all three were substantially blocked and unblocked in500-nm and 380-nm light respectively (FIG. 1a ). FIG. 1b shows the PALapproach, designed to employ a photoswitchable pore blocker tethered toendogenous amino acids on the external channel surface. After affinitylabeling the extended trans isomer presents the ammonium ion to blockthe pore while the cis isomer is too short to reach, allowing ionconduction. FIG. 1c shows photochromic blockers of the internal QAbinding site. The elongated trans isomer can fit into the innervestibule for the ammonium ion to block open channels while the bent cisisomer is not sterically accommodated and is unable to block.

Similarly, there was substantially no correlation between external TEAaffinity and AAQ effectiveness observed in a screen of differentmammalian K⁺ channels that are present in neurons. Furthermore,inclusion of TEA at concentrations that result in about 80% poreoccupancy did not produce significant changes in the apparent rate oflabeling by AAQ.

To determine whether the electrophile is a necessary component of AAQ,EtAcAQ was prepared (FIG. 2a ), which is sterically similar to AAQ butlacks the electrophilic double bond. Under the same pre-treatmentconditions, AcAQ produced considerable sustained photoswitching that isqualitatively indistinct from that obtained with AAQ (FIGS. 2b and 2c ).FIGS. 2b and 2c show voltage-gated steady-state currents from Shaker IRchannels after blocker treatment. Cells treated with either 400 uM AAQ(FIG. 2b ) or 400 uM EtAcAQ (FIG. 2c ) were exposed to 500-nm and 380-nmlight as indicated. Voltage-gated K+ currents were elicited by pulsingfrom −70 to +40 mV for 200 ms at is intervals in whole cell voltageclamp.

Like AAQ, EtAcAQ persistently blocked the entire steady-state current ina light-dependent fashion. This result provides further evidence thatAAQ does not substantially covalently attach to Shaker. Instead, itsuggests that molecules of this type represent a new class ofphotochromic ligands that can stably associate with the channel or thesurrounding environment to afford persistent block, effectivelymimicking covalent modification.

AAQ Blocks the Internal TEA Binding Site

Potassium channels are not only blocked by alkyl ammonium ions like TEAat the external entrance to the selectivity filter, but also within theinner vestibule at the internal entrance to the selectivity filter,where they bind with 1 to 2 orders of magnitude greater affinity. TheN-terminal peptide of many K+ channels also binds and blocks at thissite in a process known as fast-inactivation. Genetic deletion of theN-terminus from Shaker K+ channels produces channels that do not rapidlyinactivate and are therefore called Sh-IR (Shaker-Inactivation Removed).Many intracellular pore blockers, including long-chain alkyl ammoniumions, mimic fast-inactivation when applied to Sh-IR and othernon-fast-inactivating channels. This is observed in the current responseto a prolonged step depolarization, which opens the voltage gates toallow K+ conductance. After the rapid rising phase of channel opening,current quickly decays or “fast-inactivates” over tens of milliseconds.Because block does not occur until the inner gates have begun to open,which provides access to the inner vestibule, this phenomenon isreferred to as “open channel block.”

To explore the mechanism of AAQ action on voltage-gated K+ channels,studies were undertaken in HEK293 cells expressing Sh-IR. FIG. 3A showsthe current response of an AAQ-treated cell to depolarizing voltagesteps. Under 380 nm irradiation, channels are not blocked by AAQ andchannel opening is followed only by slow C-type inactivation (purpletrace). However, when channels are blocked by AAQ with 500 nm light,substantial peak current remains (I_(pk)), which rapidly decays so thatnearly all of the steady-state current (I_(ss)) is blocked (greentrace). This observed fast inactivation is consistent with open channelblock at the internal TEA binding site by AAQ. In contrast,fast-inactivation is not observed during blockade of SPARK channels(FIG. 3B), consistent with action at the external TEA binding site.

Positively charged intracellular K+ channel blockers typically exhibitvoltage-dependent block, such that I_(ss) is blocked more effectively atdepolarized membrane potentials. Because the internal TEA binding siteis within the membrane electric field, just as increased depolarizationincreases the driving force for positively charged K+ ions to flow inthe outward direction, alkyl ammonium ions, which cannot permeate, aredriven more tightly into the pore and therefore block more effectively.Consistent with this, block by AAQ is distinctly voltage-dependent, suchthat a higher degree of steady-state current block is observed at moredepolarized membrane potentials (FIG. 3C). When Sh-1R channels arecompletely blocked at 500 nm, the lack of current at all membranepotentials obscures any trend (FIG. 3C, green line; solid squares).However, illumination at 420 nm, which produces 50% cis isomer andtherefore ˜50% block, reveals voltage-dependent block (FIG. 3C, blueline; solid circles), as indicated by the decline in I_(ss) at moredepolarized potentials. However, under 380 nm illumination, channels arecompletely unblocked and the current increases with voltage in a linearfashion (FIG. 3C, purple line; solid triangles).

A more direct assay for open channel block is to apply the blockingagent while keeping the channels closed at a negative membranepotential. If the channels must be open before the molecules can bind tothe pore, there should be little effect on I_(pk) at the onset of thefirst opening in the presence of blocker. However, once the channels areopened by depolarization, blockade can rapidly accumulate as the innervestibule of an increasing number of channels is invaded by the blocker.To test for this mode of action, current was monitored before and afterapplication of AAQ to closed channels (FIG. 3D). After measuring theinitial current, cells were clamped at −70 mV to ensure a very lowprobability of opening and then exposed to 300 μM AAQ in the dark for 3to 4 minutes, which typically produces >70% block of the Iss whilechannels are opened at 1 Hz. After washout for 1 minute, the I_(pk)measured during the first opening was not reduced, although I_(ss)(observed 200 ms later) was slightly reduced (FIG. 3D inset). Duringsubsequent openings block rapidly accumulated and was completelyreversed upon exposure to 380 nm light. Similar results were obtained in4 other cells.

As implied by the open channel block mechanism, inhibition of voltagegated ion channels by internal blockers occurs in an activity-dependent,accumulative fashion. Functionally, this is observed as a greater degreeof block of both I_(pk) and I_(ss) when channels are opened at higherfrequencies, which decreases the recovery time between openings. To testfor activity-dependent block, cells were given depolarizing pulses atfrequencies ranging from 0.125 Hz to 2 Hz with and without exposure toAAQ. Normalized I_(pk) from two different cells is shown in FIG. 3E.Compared to 0.125 Hz, increasing the frequency of channel opening to 2Hz reduced Ipk in AAQ-treated cells by 55+/−22% (n=5) (black squares).In contrast, the I_(pk) in untreated cells was only reduced by4.5+/−0.8% (n=3) (open squares), which is likely due to accumulation ofC-type inactivation. Further confirmation of activity-dependent blockwas obtained in a related experiment, which demonstrated that recoveryfrom block correlates with the lime channels are held closed betweenopenings.

Permeant ions are known to enhance the exit rate of charged blockers,which is thought to result from electrostatic repulsion between the ionand the blocking particle. The effect of changing external K+concentration ([K+]_(o)) on the degree of block afforded by AAQ wasexamined during whole cell recordings under 500 nm illumination (FIG.3F). After establishing voltage clamp in standard external buffer([K+]_(o)=1.5 mM), cells were locally perfused with solutions containing0.3 mM and 20 mM [K+]_(o) (ionic strength and Cl⁻ concentration wereheld constant by adjusting [NaCl]). To normalize for the effects ofchanging [K+]_(o) on K+ driving force, currents were also measured at380 nm, which completely unblocks the channels. Analysis of the currentsobtained at +40 mV (normalized to 380 nm) reveals that AAQ block isinversely correlated with [K+]_(o), strongly supporting that thequaternary ammonium ion of AAQ blocks at the internal TEA binding site.

FIGS. 3A-F. AAQ is an open-channel blocker of the Sh-IR internalTEA-binding site. (a,b) Whole-cell current responses to 200 msdepolarizing current steps from −70 to +40 mV in the presence of 380 nm(purple) or 500 nm (green) light. (a) AAQ imparts fast-inactivation onSh-IR. (b) Fast-inactivation is absent in MAQ-treated SPARK channels.(c) Steady-state conductance vs. voltage curves under 380 nm (purple;solid triangles), 420 nm (blue; solid circles) and 500 nm (green; solidsquares) illumination, constructed from IN curves and normalized to thecurrent measured at 380 nm and +60 mV. (d) AAQ does not block Sh-IRuntil channels are opened by depolarization. I_(ss) measured over timeunder the indicated conditions. Channels were opened at 1 Hz. Insetshows the whole-cell current records at the indicated time points. Blackbar: −70 mV holding potential; orange bar: 300 μM AAQ; purple bar: 380nm; green bar: 500 nm. (e) Frequency-dependence of AAQ block. NormalizedI_(pk) at the indicated frequencies in a control cell (white squares)and an AAQ-treated cell (black squares). (f) Dependence of AAQ block onexternal [K+]. Summary of steady-state current responses of a cell todepolarizing current steps from −70 to +40 mV in the presence of 380 nm(purple) or 500 nm (green) light, normalized to the 380 nm response ateach [K+]_(o).

Covalent Modification is not Required

Based on these data, it was reasoned that direct application of AAQ tothe cystosol during whole cell recordings should also photosensitizechannels, possibly in a covalent fashion. When 1 mM AAQ was added to therecording pipette solution, it was found to photoregulate K+ channels.Again, blockade exhibited the fast-inactivation associated with openchannel block at the internal TEA binding site.

To further explore the interaction between AAQ and the internal TEAbinding site, inside-out patches were pulled from HEK293 cellsexpressing Sh-IR, allowing one to apply AAQ directly to the internal TEAbinding site and then wash it out. Surprisingly, current block by AAQwas relieved within several seconds of washout with control solution,indicating that covalent reaction did not occur and that AAQ may behaveas a photochromic ligand (PCL) rather than a photoswitchable tetheredligang (PTL) under these conditions. A dose-response curve was thereforegenerated for both trans and cis isomers by illuminating patches with380 and 500 nm light in the presence of different concentrations of AAQ.These experiments confirmed that AAQ can indeed block as a PCL at theinternal TEA binding site (trans-AAQ IC₅₀=2.0+/−0.21.1M, cis-AAQIC₅₀=64+/−2.1 μM, n=3-5).

These results not only establish AAQ as an internal blocker, but alsodemonstrate that it does not need to form a covalent association withthe channel to impart photosensitivity. Consistent with this notion, itis difficult to account for the observed fast inactivation andactivity-dependent block if AAQ were to attach at a site within thechannel lumen, in which case access to the internal TEA binding siteshould not depend on opening of the intracellular gates.

To determine whether covalent modification is necessary to persistentlyphotosensitize cells under conditions of external application, AAQ wasmodified. To this end, the AAQ analogue AcAQ (4) was prepared, which issterically similar to AAQ but lacks the electrophilic double bond andcannot bind covalently. Under extracellular pre-treatment conditions,AcAQ produced considerable sustained photoswitching that isqualitatively indistinct from that obtained with AAQ. Current responsesto step depolarization recorded from AcAQ-treated cells are very similarto the AAQ trace in FIG. 3A. Like AAQ, AcAQ persistently blocked thesteady-state current in a light-dependent fashion although it cannotcovalently modify channels.

Structure-Activity Relationships

These results support an intracellular mode of action for AAQ, implyingthat it at least partially crosses the cellular membrane in order toblock potassium channels. Although this seems counter-intuitive whenconsidering the permanently charged ammonium ion, a substantial numberof quaternary local anesthetics have been observed to cross cellularmembranes to reach an internal binding site on sodium and calciumchannels. Similarly, amphipathic tetraalkyl ammonium ions such as C9-TEAand C12-TEA have been observed to block voltage-gated potassium channelsinternally when applied externally at sufficiently high concentrations.AAQ and EtAcAQ are both amphipathic molecules that contain a positivelycharged hydrophilic tetraalkyl ammonium head group and a relativelyhydrophobic para-substituted azobenzene tail.

To explore the contribution of hydrophobicity to the potency ofmolecules like AAQ and AcAQ, a series of Azo-QAs was prepared (FIG. 4).The triethylammonium head group bridged to a 4-aminoazobenzene wasconserved while the 4′-substituents were replaced with aliphatic “tails”of increasing hydrophobicity (4-8). The symmetric analogue 9 was alsoprepared, as this compound would be expected to exhibit poor membranepenetration due to the presence of two positive charges. Finally theamide “tail” was either removed completely (10) or replaced with apropyl group (11) to mimic the length of AcAQ, the shortest analoguepreviously examined. Prior to screening, U V/V is spectroscopy was usedto confirm efficient photoisomerization of each AzoQA. In all cases,exposure to 380 nm and 500 nm light produced photostationary statesconsisting of at least 80% cis and trans isomers respectively.

These AzoQA's were applied in dark, which favors the transconfiguration, at various concentrations to HEK293T cells expressingSh-IR, followed by washout and whole-cell voltage clamp recording.Because these conditions did not permit measurement of the initialcurrent, we looked for the fast-inactivation associated with openchannel block to determine whether or not a molecule was able to blockthe channel. To quantify the relative potency of each new analogue, weidentified the minimal dose that was able to block >95% of I_(ss)measured under 380 nm illumination, which typically resulted in completeunblock as judged by a lack of fast inactivation.

The data obtained for the new analogues, including AcAQ (4), aresummarized in FIG. 4. Strikingly, each of the compounds containing aneutral hydrophobic tail (4-8, 10, 11) was able to persistently blockthe channels for at least several minutes of recording when applied atsufficiently high concentrations. Only compound 9 was not observed toblock at all after extracellular treatment, even at 2 mM, althoughdirect cytosolic application did afford block. A general trend wasobserved in which potency is correlated with tail length andhydrophobicity. Strikingly, compound 8, named BzAQ (Benzoyl-Azo-QA) wasfound to block >95% of I_(ss) at only 25 μM. In contrast, the truncatedanalogue 10, which lacks an extended hydrophobic tail, exhibitedessentially equal block in both cis and trans forms. This indicates thatinteractions between the “tail” and channel protein account for thedifferences in affinity between each isomer. Further supporting thisidea, we observed that compound II, named PrAQ (Propyl-Azo-QA)preferentially blocks in the cis form, which stands in contrast to allof the amide-derivatives. Although at higher concentrations, some blockby the trans isomer was also apparent as judged by the residual fastinactivation at 500 nm, 40 nM 11 provided 50% block of I_(ss) in the cisform without producing obvious block by the trans isomer.

Intracellular Application Affords Photocontrol in Individual Cells

BzAQ was applied to dissociated hippocampal cultures, a preparation inwhich AAQ was previously found to strongly photosensitize endogenous K⁺after preincubation at 300 nM. IV curves from neurons recorded inwhole-cell voltage clamp showed that steady state current is modulatedsignificantly after extracellular treatment with 20 μM BzAQ (FIG. 5A).From this data, I_(ss) at +30 mV was compared at 380 and 500 nm,revealing that on average 57+/−6.6% of I_(ss) is blocked by BzAQ underthese conditions. In current clamp mode, which allows observation ofaction potentials, BzAQ was observed to significantly depolarize thecellular membrane potential when switched from cis to trans, which wassufficient to induce action potential firing when the cell was nearthreshold (FIG. 5B). Together, these data indicate that BzAQ canadequately substitute for AAQ, but with the advantage of increasedpotency.

FIGS. 5A and 5B. BzAQ photoregulates endogenous K+ channels indissociated hippocampal neurons to modulate neural activity. (a)Steady-state current vs. voltage curves under 380 nm (purple; solidcircles) and 500 nm (green; solid squares) illumination recorded fromneurons treated with 20 nM BzAQ. Recordings from individual cells werenormalized to the current measured at 380 nm and +30 mV. (b) Currentclamp recording from a neuron showing the induction of action potentialfiring in response to 500 nm light.

Example 2 Synthesis of Synthetic Regulators

General Synthetic Methods.

Reactions were carried out under N₂ atmosphere in flame dried glassware.Tetrahydrofuran (THF) was distilled from Na/benzophenone immediatelyprior to use. Acetonitrile (MeCN), and diisopropylethylamine (DIPEA)were distilled from CaH₂ immediately prior to use. All other reagentsand solvents were used without further purification from commercialsources. Flash column chromatography was carried out with EcoChrom ICNSiliTech 32-63 D 60 Å silica gel. Reverse-phase chromatography wascarried out with Waters Preperative C18 Silica Gel WAT010001 125 Å andWaters Sep-Pak Vac 20 cc C18 Cartridges WAT036925. Reactions andchromatography fractions were monitored with either Merck silica gel60F254 plates or Analtech C18 silca gel RPS-F 52011 plates, andvisualized with UV light and 0.1N HCl. NMR spectra were measuredspecified solvents and calibrated from residual solvent signal on aBruker DRX spectrometer at 500 MHz for ¹H spectra and 125 MHz for ¹³Cspectra and either a Bruker AVB or Bruker AVQ spectrometer at 400 MHzfor ¹H spectra and 100 MHz for ¹³C spectra. UV/VIS spectra were measuredin water at a concentration of 10 nM on an Agilent 8453 UV/VISspectrometer. Photoisomerization in solution was achieved by irradiationwith the un-collimated quartz fiber optic output from a Polychrome Vmonochromator (Till Photonics).

AAQ (6) was synthesized as previously described (Fortin et al. (2008)Nature Methods 5:331) and compounds 7-14 were prepared according to thesame general procedures (Scheme 1, below), which are described brieflybelow. 4,4′-diaminoazobenzene and 4-aminoazobenzene were purchased fromcommercial sources while compound S6 was prepared from following thegeneral procedure of Priewisch and Ruck-Braun (2005) J. Org. Chem.70:2350, as described below.

General Procedure for Mono-Acylation of 4,4′-Diaminoazobenzene.

To a solution of 4,4′-diaminoazobenzene (1 eq) and DIPEA (1.2 eq) in THFat 0° C. was added the acid chloride (1.0 eq) in THF over 1 h. Thereaction was stirred for 15 min, warmed to room temperature and stirredfor 1 h, at which time the crude mixture was removed of solvent in vacuoand immediately dry loaded onto silca gel (2 g) for chromatography.

4-acetamido-4′-aminoazobenzene (S1)

Following the general monoacylation procedure, 4,4′-azodianiline (25 mg,120 μmol) was treated with acetyl choloride (8.5 μl, 120 μmol). Silicagel chromatography through a wide column (20% ethyl acetate indichloromethane, gradient to 66%) provided S1 as an orange solid (22 mg,90 μmol, 73% yield): ¹H (CD₃CN, 500 MHz): 1.98 (s, 3H); 4.77 (s, 21-1);6.74 (d, 2H, J=8.5); 7.70 (d, 4H. J=8.5); 7.77 (d, 21-1, J=8.5); 8.53(s, 1H). ¹³C(CD₃CN, 125 MHz): 23.4, 113.9, 119.3, 122.7, 124.7, 140.6,144.3, 148.6, 151.5, 168.7. HRMS (FAB+): calc'd for C₁₄H₁₄N₄O—254.1168.found—254.1171 (M+).

4-propionamido-4′-aminoazobenzene (S2)

Following the general monoacylation procedure, 4,4′-azodianiline (50 mg,240 μmol) was treated with propionyl choloride (21 μl, 240 μmol). Silicagel chromatography through a wide column (50% ethyl acetate in hexanes,gradient to 66%) provided S2 as an orange solid (43 mg, 162 μmol, 68%yield): ¹H (MeOD, 500 MHz): 1.23 (t, 31-1, J=7.5); 2.41 (q, 21-1,J=7.5); 6.73 (d, 2H, J=8.5); 7.69 (d, 4H, J=8.5); 7.75 (d, 2H, J=8.5).¹³C (MeOD, 125 MHz): 8.73, 29.64, 113.8, 199.7, 122.3, 124.5, 134.0,144.3, 149.0, 151.8, 174.0. HRMS (ESI+): calc'd for C₁₅H₁₇N₄O⁺-269.1397.found 269.1400 (MH+).

4-butyramido-4′-aminoazobenzene (S3)

Following the general monoacylation procedure, 4,4′-azodianiline (50 mg,240 μmol) was treated with butyryl choloride (25 μl, 240 μmol). Silicagel chromatography through a wide column (50% ethyl acetate in hexanes,gradient to 66%) provided S3 as an orange solid (44 mg, 157 μmol, 65%yield): ¹H (MeOD, 400 MHz): 1.01 (t, 3H, J=7.6); 1.74 (q, 21-1, J=7.6);2.38 (t, 2H, J=7.6); 6.73 (d, 2H, J=8.8); 7.69 (d, 4H, J=8.8); 7.77 (d,2H, J=8.8). ¹³C (MeOD, 100 MHz): 14.2, 20.4, 40.1, 115.4, 121.2, 121.3,123.9, 124.7, 126.1, 141.5, 142.8, 145.9, 150.7, 153.4, 174.8. HRMS(FAB+): calc'd for C₁₆H₁₉N₄O⁺-283.1559. found-283.1556 (MH+).

4-(pent-4-en)amido-4′-aminoazobenzene (S4)

Following the general monoacylation procedure, 4,4′-azodianiline (50 mg,240 μmol) was treated with 4-pentenoyl choloride (27 μl, 240 μmol).Silica gel chromatography through a wide column (5% ethyl acetate indichloromethane, gradient to 75%) provided S4 as an orange solid (48 mg,163 μmol, 68% yield): ¹H (MeOD, 500 MHz): 2.34-2.42 (m, 4H); 4.89-5.03(m, 21-1); 5.72-5.86 (m, 1H); 6.63 (d, 2H, J=8.6); 7.58 (d, 4H, J=8.6);7.65 (d, 2H, J=8.6). ¹³C (MeOD, 125 MHz): 29.3, 35.9, 113.8, 114.5,119.7, 122.3, 124.5, 136.7, 139.8, 144.3, 149.1, 151.8, 172.4. HRMS(FAB+): calc'd for C₁₇H₁₈N₄O—294.1481. found—294.1487 (M+).

4-benzamido-4′-aminoazobenzene (S5)

Following the general monoacylation procedure, 4,4′-azodianiline (50 mg,240 μmol) was treated with benzoyl choloride (26 μl, 240 μmol). Silicagel chromatography through a wide column (10% ethyl acetate indichloromethane) provided S5 as an orange solid (44 mg, 130 μmol, 54%yield): ¹H (DMSO, 400 MHz): 6.04 (s, 2H); 6.66 (d, 2H, J=8.4); 7.52-7.65(m, 5H); 7.76 (d, 2H, J=8.8); 7.88-7.98 (m, 4H); 10.46 (s, 1H). ¹³C(DMSO, 100 MHz): 113.4, 120.5, 122.3, 124.9, 127.7, 128.4, 131.7, 134.8,140.4, 142.9, 148.4, 152.5, 165.7. HRMS (ESI+): calc'd forC₁₉H₁₇N₄O⁺—317.1397. found—317.1403 (MH+).

4-amino-4′-propylazobenzene (S6)

To a solution of 4-propylaniline (541 μl, 3.7 mmol) in dichloromethane(12.5 ml) was added a solution of oxone (4.55 g, 7.4 mmol) in H₂O (50ml). The biphasic mixture was stirred vigorously under nitrogen for 1 h,at which time the color had become deep aqua green and the reaction wasjudged as complete by TLC (1:1 hexanes:ethyl acetate; Rf=0.83). Themixture was transferred to a separatory funnel and the organic layer wasremoved. The aqueous layer was extracted twice with dichloromethane (20ml). The combined organics were subsequently washed with 20 ml each of1M HCl, sat. NaHCO₃, H₂O, and brine and then dried over Na₂SO₄, filteredand removed of solvent in vacuo to provide nitrosyl-4-propylbenzene as agreen oil. The crude product was immediately dissolved in glacial aceticacid (30 ml) and stirred under nitrogen, followed by the addition of1,4-diaminobenzene (400 mg, 3.7 mmol) in dry DMSO (10 ml). The reactionwas allowed to stir for 48 hrs, during which time the color turnedorange-brown. The mixture was transferred to a separatory funnelcontaining 100 ml brine and extracted five times with ethyl acetate (50ml). The combined organics were then washed with dilute NaCl (50 ml) sixtimes to remove the DMSO and then dried of Na₂SO₄, filtered removed ofsolvent in vacuo. Silica gel chromatography through a wide column (40%ethyl acetate in hexanes) provided S6 as an orange solid (432 mg, 1.8mmol, 49% yield): ¹H (MeOD, 500 MHz): 0.95 (t, 3H, J=7.5); 1.66 (q, 2H,J=7.5); 2.62 (t, 2H, J=7.5); 6.74 (d, 2H, J=8.5); 7.26 (d, 2H, J=8) 7.70(d, 4H, J=8.5). ¹³C (MeOD, 125 MHz): 12.7, 24.2, 37.4, 113.8, 121.7,124.6, 128.7, 144.3, 144.4, 151.2, 151.8. HRMS (FAB+): calc'd forC₁₅H₁₈N₃ ⁺—240.1495. found—240.1500 (MH+).

General Procedure for Acylation of Aminoazohenzenes with2-Triethylammonium Acetic Acid Chloride Chloride

To a solution of the aminoazobenzene (1 eq) and DIPEA (2 eq) in 1:1MeCN:DMF at 0° C. was added 2-triethylammonium acetic acid chloridechloride⁸ (1.5 eq) in 1:1 MeCN:DMF and stirred for 15 min, then warmedto ambient temperature and stirred for 1-12 h at which time the solventwas removed in vacuo for purification by reverse phase silica gelchromatography (0.1% formic acid in H₂O, gradient up to 50% MeCN: 0.1%formic acid in H₂O).

4-acetamido-4′-(2)-triethylammoniumacetamidoazobenzene formate; AcAQ (7)

Following the general acylation procedure,4-acetamido-4′-aminoazobenzene (S1) (10 mg, 39 μmol) provided 7 as anorange solid (15.9 mg, 36 μmol, 93% yield): ¹H (MeOD, 500 MHz): 1.39(bs, 9H); 2.17 (s, 3H); 3.68 (bs, 6H); 4.23 (s, 2H); 7.74-7.91 (m, 8H);8.54 (bs, 1H). ¹³C (MeOD, 125 MHz): 6.5, 22.6, 54.4, 56.1, 119.6, 120.0,123.2, 139.8, 141.4, 148.6, 149.3, 161.8, 170.4, 181.6. HRMS (ESI+):calc'd for C₂₂H₃₀N₅O₂ ⁺—396.2394. found—396.2393 (M+).

4-propionamido-4′-(2)-triethylammoniumacetamidoazobenzene formate (8)

Following the general acylation procedure,4-propionamido-4′-aminoazobenzene (S2) (10 mg, 37 μmol) provided 8 as anorange solid (9.2 mg, 20 μmol, 55% yield): ¹H (MeOD, 500 MHz): 1.22 (t,3H, J=7.5); 1.39 (t, 911, J=7.5); 2.43 (q, 2H, J=7.5); 168 (q, 6H,J=7.5); 4.20 (s, 2H); 7.75-7.80 (m, 4H); 7.86-7.91 (m, 4H); 8.44 (bs,1H). ¹³C (MeOD, 125 MHz): 6.5, 8.7, 29.7, 54.3, 54.4, 56.1, 119.6,120.0, 123.2, 139.7, 141.6, 148.5, 149.4, 161.7, 174.1, 181.5. HRMS(ESI+): calc'd for C₂₃H₃₂N₅O₂ ⁺—410.2551. found—410.2548 (M+).

4-butyramido-4′-(2)-triethylammoniumacetamidoazobenzene formate (9)

Following the general acylation procedure,4-butyramido-4′-aminoazobenzene (S3) (10 mg, 35 μmol) provided 9 as anorange solid (8.7 mg, 18.5 μmol, 53% yield): ¹H (MeOD, 500 MHz): 1.01(t, 3H, J=7.5); 1.39 (t, 911, J=7.5); 1.74 (q, 2H, J=7.5); 2.39 (t, 2H,J=7.5); 3.68 (q, 6H, J=7.5); 4.18 (s, 2H); 7.75-7.80 (m, 4H); 7.87-7.92(m, 4H); 8.55 (bs, 1H). ¹³C (MeOD, 125 MHz): 6.5, 12.5, 18.8, 38.5,54.3, 119.6, 120.0, 123.2, 139.7, 141.5, 148.6, 149.4, 161.7, 173.3,181.6. HRMS (ESI+): calc'd for C₂₄H₃₄N₅O₂ ⁺—424.2707. found—424.2704(M+).

4-(pent-4-en)amido-4′-(2)-triethylammoniumacetamidoazobenzene formate(10)

Following the general acylation procedure,4-(pent-4-en)amido-4′-aminoazobenzene (S4) (10 mg, 34 μmol) provided 10as an orange solid (13 mg, 27 μmol, 79% yield): ¹H (MeOD, 500 MHz): 1.38(t, 9H, J=7.5); 2.44-2.51 (m, 4H); 3.67 (q, 6H, J=7.5); 4.22 (s, 2H);5.01 (d, 2H, J=10); 5.10 (d, 2H, J=17); 5.85-5.93 (m, 1H); (7.74-7.80(m, 4H); 7.86-7.90 (m, 4H); 8.33 (bs, 1H). ¹³C (MeOD, 125 MHz): 6.5,29.2, 35.9, 54.4, 56.1, 114.5, 119.6, 120.0, 123.2, 136.7, 139.8, 141.4,148.6, 149.3, 161.8, 172.5. HRMS (ESI+): calc'd for C₂₅H₃₄N₅O₂⁺—436.2707. found—436.2702 (M+).

4-benzamido-4′-(2)-triethylammoniumacetamidoazobenzene formate; BzAQ(11)

Following the general acylation procedure,4-benzamido-4′-aminoazobenzene (S5) (10 mg, 32 μmol) provided 11 as anorange solid (6 mg, 12 μmol, 38% yield): ¹H (MeOD, 400 MHz): 1.41 (t,9H, J=7.5); 3.69 (q, 6H, J=7.5); 4.21 (s, 2H); 7.52-7.63 (m, 3H); 7.81(d, 2H, J=8.8); 7.93-7.98 (m, 8H); 8.47 (bs, 1H). ¹³C (MeOD, 100 MHz):6.5, 54.3, 120.0, 120.6, 123.1, 123.2, 127.3, 128.2, 131.7, 134.7,139.8, 141.5, 148.9, 149.4, 167.5. HRMS (ESI+): calc'd for C₂₇H₃₂N₅O₂⁺—458.2551. found—448.2550 (M+).

4-4′-bis[(2)-triethylammoniumacetamido]azobenzene bis-formate (12)

Following the general acylation procedure, 4,4′-diaminoazobenzene (10mg, 47 μmol) was treated with 141 μmol 2-triethylammonium acetic acidchloride chloride to provide 12 as an orange solid (20 mg, 34 μmol, 73%yield): ¹H (MeOD, 500 MHz): 1.39 (t, 18H, J=7.5); 3.68 (q, 12H, J=7.5);4.24 (s, 4H): 7.81 (d, 4H. J=8.5); 7.92 (d, 4H, J=8.5); 8.41 (bs, 3H).¹³C (MeOD, 125 MHz): 6.5, 54.4, 56.1, 120.1, 123.3, 140.1, 149.3, 161.9,181.5. HRMS (ESI+): calc'd for C₂₈H₄₄N₆O₂ ^(z2+)—248.1757.found—248.1756 (M2+/2).

4-(2)-triethylammoniumacetamidoazobenzene formate (13)

Following the general acylation procedure, 4-aminoazobenzene (10 mg, 51μmol) provided 13 as an orange solid (13.4 mg, 35 μmol, 69% yield): ¹H(MeOD, 500 MHz): 1.39 (t, 9H, J=7.5); 3.68 (q, 6H, J=7.5); 4.22 (s, 2H);7.48-7.55 (m, 3H); 7.81 (d, 2H, J=8.5); 7.89 (d, 2H, J=7.5); 7.93 (d,2H, J=8.5); 8.42 (bs, 1H). ¹³C (MeOD, 125 MHz): 6.5, 54.3, 56.1, 120.0,122.3, 123.4, 128.8, 130.7, 140.1, 149.2, 152.5, 161.9. HRMS (ESI+):calc'd for C₂₀H₂₇N₄O⁺—339.2179. found—339.2178 (M+).

4-propyl-4′-(2)-triethylammoniumacetamidoazobenzene formate; PrAQ (14)

Following the general acylation procedure, 4-amino-4′-propylazohenzene(S6) (10 mg, 42 μmol) provided 14 as an orange solid (10.3 mg, 24 μmmol,57% yield): ¹H (MeOD, 500 MHz): 0.98 (t, 3H, J=7.5); 1.40 (t, 9H,J=7.5); 1.70 (q, 2H, J=7.5); 2.68 (t, 2H, J=7.5); 3.68 (q, 6H, J=7.5);4.21 (s, 2H); 7.36 (d, 2H, J=8.5); 7.81 (t, 4H, J=8.5); 7.92 (d, 4H,J=8.5); 8.51 (bs, 1H). ¹³C (MeOD, 125 MHz): 6.5, 8.7, 29.7, 54.3, 54.4,56.1, 119.6, 120.0, 123.2, 139.7, 141.6, 148.5, 149.4, 161.7, 174.1,181.5. HRMS (ESI+): calc'd for C₂₃H₃₃N₄O—381.2649. found—381.2650 (M+).

Example 3 Characterization of QAQ

Experiments were conducted to test the effect of QAQ on variouschannels. It was found that QAQ is able, like local anesthetic, to blockvoltage gated K, Na and Ca channels, to a similar extent. QAQ was shownto photoregulate Shaker K channel and also other K channel subtypes suchas Kν2, Kν3 Kν4 and native K channels in hippocampal cells. QAQ was alsoshown to photosensitizes native Na channels expressed in NG108-15 cells.QAQ was further shown to photosensitize L type and N type Ca channels.

For all those channels (Kν2.1, Kν3.1, Kν4.2, hippocampal Kν, Naν, L-typeCaν, and Caν2.2) the block occurred in the dark, or under 500 nm light.Purple light was used in all cases to relieve the block. FIG. 6 depictsa bar graph showing the quantification of photoswitching on individualchannels when having 100 μM QAQ in the pipette.

The effect of QAQ on neurons was investigated. Neuron firing wasexamined under current clamp, by injecting progressively greater currentinto the cell, under 2 different wavelengths of light. As shown in FIG.7, under green light, the cell does at best one action potential (AP),but remains silent, regardless of the current injected. In contrast,under purple light, the cell fires normally. This is because block of Nachannels dominates: when Na channels are blocked, the initial phase ofthe AP is blocked and neurons are silent. Again, QAQ acts like lidocainein neurons, silencing cells under green light or in the dark.

QAQ was found to be membrane impermeant. When 100 μM QAQ was appliedthrough the patch pipette to HEK cells expressing Shaker channel,photoregulation of the K channel current was observed. The K current wasobserved upon a depolarization to +40 mV under purple 380 nm light.Applying green light resulted in a dramatic decrease in K current. Incontrast, no photoregulation of K current was seen when QAQ wasexternally applied, showing that QAQ does not cross the cell membrane.

Advantage was taken of the biophysical properties of certain ionchannels. TRPV1 channel and the ATP gated P2X7 receptor can open a widepore upon prolonged agonist exposure. This pore becomes then permeableto large organic cations like the local anesthetic QX314. It wasinvestigated whether QAQ could be loaded into cells expressing eitherone of these two channels (P2X₇R or TRPV₁). If so, specific cells couldbe targeted with QAQ, and then excitability could be controlled withlight. This concept is depicted schematically in FIG. 8.

It was first determined whether P2X₇R expressed in HEK cells could be anentry route for QAQ. P2X₇R was co-expressed together with Shakerchannel. As shown in FIG. 9, QAQ could be loaded in the HEK cell only ifP2X7 channels were expressed and ATP, the agonist for the P2X₇ channels,was coapplied together with QAQ. As shown in FIG. 9, photoswitchingoccurred in cells coexpressing P2X₇R and Shaker when QAQ was loaded intothe cells in the presence of ATP. Even a short application of 5 min wasenough to get very good photoswitching (light-controlled activation) ofShaker channels current. Also as shown in FIG. 9, QAQ could be loadedinto cells co-expressing TRPV₁ and Shaker channel in the presence of theTRPV₁ agonist capsaicin. Furthermore, when cells co-expressing TRPV₁ andShaker channel were loaded with QAQ (in the presence of capsaicin),photoswitching could be effected.

Hippocampal neurons were transfected with P2X₇R. As shown in FIG. 10(left panel) QAQ could be loaded into cells only when P2X7 receptorswere expressed. In 1 of 13 cells tested, QAQ entered a cell which wasnot heterologously expressing P2X₇R, possibly because this cell wasexpressing endogenous P2X₇ receptors. It was then asked whether QAQcould enter cells naturally expressing either of these channels andlooked at sensory neurons. Sensory neurons are activated by sensoryinput and project towards the central nervous system. Sensory neurons ofthe trigeminal ganglia naturally express TRPV1 channels. QAQ was appliedtogether with capsaicin on these sensory neurons in in vitro culture.Again, it very robust photoswitching of native K channels was seen (FIG.10, right panel).

Example 4 Photoregulationg Nociception In Vivo

It was shown (Example 3) that QAQ blocks capsaicin-evoked sensory neuronactivation in vitro, in a light-dependent manner. It was then testedwhether QAQ can block nocifensive responses in vivo, and whether thisaction can be regulated by light. This experiment consisted of 2 parts:(1) it was tested whether QAQ acts as an analgesic, in the absence oflight. In vitro studies suggested that in ambient light, QAQ is in anactive form and thus should block nocifensive behaviors by blockingactivity of C-fibers; (2) based on the results of part (1), the sameexperiment was performed in the presence of 380 nm light.

Injection of capsaicin into the mouse hindpaw elicits a 5-10 min bout oflicking and flinching; such responses are blocked by co-application oflidocaine, morphine, and other analgesics (Binshtock et al., 2007 Nature449:607-10). Because analgesics are molecules that alleviate pain,evaluating the analgesic properties of a new molecule requires inductionof discomfort in animals. The ability of QAQ to inhibit capsaicin-evokednocifensive behaviors was tested. Fourteen mice were used for thisexperiment: to one set of 7 mice, capsaicin alone was applied byinjection; to a second set of 7 mice, capsaicin and QAQ wereco-injected. The mice were then placed in a transparent Plexiglaschamber and behavior was video recorded for 10 min. After 10 min, micewere euthanized. As shown in FIG. 11, co-injection of QAQ along withcapsaicin reduced the number of licks and the time spent paw lifting.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A compound having the formula:(A)_(n)-X₁-(B)_(m)-X₂-(C)_(p) wherein A is a polypeptide associationmoiety selected from hydrogen, C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl,—NR¹⁰R¹¹, —NR¹²C(O)R¹³, C₂₋₁₀ alkenyl, substituted C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, substituted C₂₋₁₀ alkynyl, C₆₋₂₀ aryl, substituted C₆₋₂₀ aryl,heteroaryl, heterocyclic, heterocyclooxy, heterocyclothio,heteroarylamino, heterocycloamino, C₄₋₁₀ cycloalkyl, substituted C₄₋₁₀cycloalkyl, C₄₋₁₀ cycloalkenyl, substituted C₄₋₁₀ cycloalkenyl, cyano,halo, —OR¹⁰, —C(O)OR¹⁰, —SR¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰; wherein R¹⁰ and R¹¹are independently selected from hydrogen and C₁₋₁₀ alkyl; R¹² ishydrogen or C₁₋₁₀ alkyl; and R¹³ is selected from hydrogen, C₁₋₁₀ alkyl,C₁₋₈ alkenyl, C₆₋₁₀ aryl, and substituted C₁₋₁₀ alkyl; B is aphotoisomerizable group comprising an azobenzene; C is a ligand selectedfrom an agonist, an antagonist, an allosteric modulator, and a blocker;n is 1; each of m and p is independently an integer from 1 to 10; and X₁and X₂ are each an optional spacer independently selected from alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, acyl, acylamino, and aminoacyl.
 2. The compound of claim 1,wherein the polypeptide association moiety comprises a group selectedfrom C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, and —NR¹⁰R¹¹, wherein R¹⁰and R¹¹ are independently selected from hydrogen and C₁₋₁₀ alkyl.
 3. Thecompound of claim 1, wherein the polypeptide association moietycomprises —NR¹⁰R¹¹, wherein each of R¹⁰ and R¹¹ are hydrogen.
 4. Thecompound of claim 1, wherein the polypeptide association moietycomprises —NR¹⁰R¹¹, wherein each of R¹⁰ and R¹¹ are C₁₋₁₀ alkyl.
 5. Thecompound of claim 1, wherein the polypeptide association moietycomprises —NR¹⁰R¹¹, wherein each of R¹⁰ and R¹¹ are methyl.
 6. Thecompound of claim 1, wherein the polypeptide association moietycomprises a substituted C₁-C₁₀ alkyl.
 7. The compound of claim 1,wherein the polypeptide association moiety comprises a C₁-C₁₀ alkyl withone or more halogen substituents.
 8. The compound of claim 1, whereinthe polypeptide association moiety comprises a C₁-C₁₀ alkyl with one ormore fluoro substituents.
 9. The compound of claim 1, wherein thepolypeptide association moiety comprises a methyl with one or morehalogen substituents.
 10. The compound of claim 1, wherein thepolypeptide association moiety comprises a methyl with one or morefluoro substituents.
 11. The compound of claim 1, wherein each of n, m,and p is one.
 12. The compound of claim 1, wherein the ligand is aligand for an ion channel.
 13. The compound of claim 12, wherein the ionchannel is a sodium channel, a potassium channel, a calcium channel, ora chloride channel.
 14. The compound of claim 1, wherein the ligand isselected from an α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid(AMPA) receptor agonist, a GABA_(A) receptor ligand, and a transientreceptor potential cation channel, subfamily V, member 1 (TRPV₁) ligand.15. The compound of claim 1, wherein the compound is a compound ofFormula XII:

wherein x is 1; y is 1; R¹ is —NR¹⁰R¹¹; R² is hydrogen; R³, R⁴, and R⁵are each independently C₂₋₈ alkyl; R⁶ is hydrogen; R¹⁰ is C₁₋₁₀ alkyl;R¹¹ is substituted C₁₋₁₀ alkyl; and or a pharmaceutically acceptablesalt thereof.
 16. The compound of claim 15, wherein: R³, R⁴, and R⁵ areeach C₃alkyl; R¹⁰ is C₂alkyl; and R¹¹ is methyl substituted with anaryl.
 17. The compound of claim 16, wherein R¹¹ is methyl substitutedwith phenyl.
 18. The compound of claim 15, wherein: R³, R⁴, and R⁵ areeach C₄alkyl; R¹⁰ is C₂alkyl; and R¹¹ is methyl substituted with anaryl.
 19. The compound of claim 18, wherein R¹¹ is methyl substitutedwith phenyl.
 20. The compound of claim 15, wherein: R³, R⁴, and R⁵ areeach C₂alkyl; R¹⁰ is C₂alkyl; and R¹¹ is methyl substituted withsubstituted aryl.
 21. The compound of claim 20, wherein R¹¹ is methylsubstituted with substituted phenyl.
 22. The compound of claim 1,wherein the compound is a compound of Formula XI:

wherein Q¹ is —C(═O)—; Q² is NR³R⁴; R³ and R⁴ are each C₂alkyl; R¹ is—NR¹⁰R¹¹; x is 1; y is 1; R² is hydrogen; R⁶ is hydrogen; R¹⁰ isC₂alkyl; and R¹¹ is methyl substituted with aryl; or a pharmaceuticallyacceptable salt thereof.
 23. The compound of claim 1, wherein: A is an—NR¹⁰R¹¹ group; R¹⁰ is C₁₋₁₀ alkyl; R¹¹ is substituted C₁₋₁₀ alkyl; B isa photoisomerizable group comprising an azobenzene; C is a ligandselected from an agonist, an antagonist, an allosteric modulator, and ablocker; each of n, m, and p is 1; and X₁ and X₂ are each an optionalspacer independently selected from alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, acyl, acylamino, andaminoacyl.
 24. The compound of claim 23, wherein R¹¹ is methylsubstituted with aryl.
 25. The compound of claim 24, wherein R¹¹ ismethyl substituted with phenyl.
 26. The compound of claim 23, whereinR¹⁰ is C₂alkyl.
 27. The compound of claim 23, wherein the ligand is aligand for an ion channel.
 28. The compound of claim 27, wherein the ionchannel is a sodium channel, a potassium channel, a calcium channel, ora chloride channel.